Polymers of isoprene from renewable resources

ABSTRACT

It has been found that certain cells in culture can convert more than about 0.002 percent of the carbon available in the cell culture medium into isoprene. These cells have a heterologous nucleic acid that (i) encodes an isoprene synthase polypeptide and (ii) is operably linked to a promoter. The isoprene produced in such a cultured medium can then be recovered and polymerized into synthetic rubbers and other useful polymeric materials. The synthetic isoprene containing polymers of this invention offer the benefit of being verifiable as to being derived from non-petrochemical based resources. They can also be analytically distinguished from rubbers that come from natural sources. The present invention more specifically discloses a polyisoprene polymer which is comprised of repeat units that are derived from isoprene monomer, wherein the polyisoprene polymer has δ 13 C value of greater than −22‰.

This application claims benefit of U.S. Provisional Patent ApplicationSer. No. 61/133,521, filed on Jun. 30, 2008. The teachings of U.S.Provisional Patent Application Ser. No. 61/133,521 are incorporatedherein by reference in their entirety.

BACKGROUND OF THE INVENTION

Isoprene (2-methyl-buta-1,3-diene) is an extremely important organiccompound that is used in a wide array of applications. For instance,isoprene is employed as an intermediate or a starting material in thesynthesis of numerous chemical compositions and polymers. Isoprene isalso an important biological material that is synthesized naturally bymany plants and animals, including humans. Isoprene is a colorlessliquid at room temperature and is highly flammable. The structuralformula of isoprene is:

Isoprene became in important monomer for utilization in the synthesis ofcis-1,4-polybutadiene when its stereo-regulated polymerization becamecommercially possible in the early 1960s. Cis-1,4-polyisoprene made bysuch stereo-regulated polymerizations is similar in structure andproperties to natural rubber. Even though it is not identical to naturalrubber it can be used as a substitute for natural rubber in manyapplications. For instance, synthetic cis-1,4-polyisoprene rubber iswidely used in manufacturing tires and other rubber products. Thisdemand for synthetic cis-1,4-polyisoprene rubber consumes a majority ofthe isoprene available in the worldwide market. The remaining isopreneis used in making other synthetic rubbers, block copolymers, and otherchemical products. For instance, isoprene is used in makingbutadiene-isoprene rubbers, styrene-isoprene copolymer rubbers,styrene-isoprene-butadiene rubbers, styrene-isoprene-styrene blockcopolymers, and styrene-isoprene block copolymers.

Over the years many synthesis routes for producing isoprene have beeninvestigated. For instance, the synthesis of isoprene by reactingisobutylene with formaldehyde in the presence of a catalyst is describedin U.S. Pat. Nos. 3,146,278, 3,437,711, 3,621,072, 3,662,016, 3,972,955,4,000,209, 4,014,952, 4,067,923, and 4,511,751. U.S. Pat. No. 3,574,780discloses another process for the manufacture of isoprene by passing amixture of methyl-tert-butyl ether and air over mixed oxide catalysts.The methyl-tert-butyl ether is then cracked into isobutylene andmethanol over the catalyst. The methanol produced is oxidized intoformaldehyde which then reacts with the isobutylene over the samecatalyst to produce the isoprene. U.S. Pat. No. 5,177,290 discloses aprocess for producing dienes, including isoprene, which involvesreacting a reaction mixture of a tertiary alkyl ether and a source ofoxygen over two functionally distinct catalysts under reactionconditions sufficient to produce high yields of the dienes with minimalrecycle of the ether.

The isoprene used in industrial applications is typically produced as aby-product of the thermal cracking of petroleum or naphtha or isotherwise extracted from petrochemical streams. This is a relativelyexpensive energy-intensive process. With the worldwide demand forpetrochemical based products constantly increasing, the cost of isopreneis expected to rise to much higher levels in the long-term and itsavailability is limited in any case. In other words, there is a concernthat future supplies of isoprene from petrochemical based sources willbe inadequate to meet projected needs and that prices will rise tounprecedented levels. Accordingly, there is a current need to procure asource of isoprene from a low cost, renewable source which isenvironmentally friendly.

SUMMARY OF THE INVENTION

It has been found that certain cells in culture can convert more thanabout 0.002 percent of the carbon available in the cell culture mediuminto isoprene. These cells have a heterologous nucleic acid that (i)encodes an isoprene synthase polypeptide and (ii) is operably linked toa promoter. In some cases, these cells are cultured in a culture mediumthat includes a carbon source, such as, but not limited to, acarbohydrate, glycerol, glycerine, dihydroxyacetone, one-carbon source,oil, animal fat, animal oil, fatty acid, lipid, phospholipid,glycerolipid, monoglyceride, diglyceride, triglyceride, renewable carbonsource, polypeptide (e.g., a microbial or plant protein or peptide),yeast extract, component from a yeast extract, or any combination of twoor more of the foregoing. The isoprene produced in such a culturedmedium can then be recovered and polymerized into synthetic rubbers andother useful polymeric materials.

It is anticipated that there will be a significant demand for syntheticrubber and other isoprene containing polymers that are synthesized usingisoprene of this type which is made from renewable, non-petrochemicalbased resources. In fact, it is believed that industrial customers andconsumers would prefer to purchase isoprene containing polymers that arederived from such environmentally friendly sources to those that aremade with isoprene derived from a petrochemical process. It is furtherbelieved that customers would be willing to pay premium prices for suchenvironmentally friendly products that are made with renewableresources. However, it is important to be able to verify that suchisoprene containing polymers are actually made from non-petrochemicalbased resources. The synthetic isoprene containing polymers of thisinvention offer the benefit of being verifiable as to being derived fromnon-petrochemical based resources. They can also be analyticallydistinguished from rubbers that come from natural sources.

The present invention more specifically discloses a polyisoprene polymerwhich is comprised of repeat units that are derived from isoprenemonomer, wherein the polyisoprene polymer has δ¹³C value of greater than−22‰. This type of polyisoprene can be a polyisoprene homopolymer.

The subject invention further reveals a polyisoprene polymer which iscomprised of repeat units that are derived from isoprene monomer,wherein the polyisoprene polymer has δ¹³C value which is within therange of −30‰ to −28.5‰. This type of polyisoprene can also be apolyisoprene homopolymer.

The present invention also discloses a polyisoprene polymer which iscomprised of repeat units that are derived from isoprene monomer,wherein the polyisoprene is free of protein, and wherein thepolyisoprene polymer has δ¹³C value which is within the range of −34‰ to−24‰.

This invention further reveals a polyisoprene polymer which is comprisedof repeat units that are derived from isoprene monomer, wherein thepolyisoprene polymer has a cis-1,4-microstructure content of less than99.9%, wherein the polyisoprene polymer has a trans-1,4-microstructurecontent of less than 99.9%, and wherein the polyisoprene polymer hasδ¹³C value of which is within the range of −34‰ to −24‰.

The subject invention also discloses a polyisoprene polymer which iscomprised of repeat units that are derived from isoprene monomer,wherein the polyisoprene polymer has a 3,4-microstructure content ofgreater than 2%, and wherein the polyisoprene polymer has δ¹³C value ofwhich is within the range of −34‰ to −24‰.

The present invention further reveals a polyisoprene polymer which iscomprised of repeat units that are derived from isoprene monomer,wherein the polyisoprene polymer has a 1,2-microstructure content ofgreater than 2%, and wherein the polyisoprene polymer has δ¹³C value ofwhich is within the range of −34‰ to −24‰.

The subject invention also discloses a polymer which is comprised ofrepeat units that are derived from isoprene monomer and at least oneadditional monomer, wherein the polymer includes blocks of repeat unitsthat are derived from isoprene, and wherein the blocks of repeat unitsthat are derived from isoprene have a δ¹³C value of greater than −22‰.

The present invention further reveals a polymer which is comprised ofrepeat units that are derived from isoprene monomer and at least oneadditional monomer, wherein the polymer includes blocks of repeat unitsthat are derived from isoprene, and wherein the blocks of repeat unitsthat are derived from isoprene have a δ¹³C value which is within therange of −34‰ to −24‰.

The subject invention also discloses a liquid polyisoprene polymer whichis comprised of repeat units that are derived from isoprene monomer,wherein the polyisoprene polymer has weight average molecular weightwhich is within the range of 5,000 to 100,000, and wherein the liquidpolyisoprene polymer has δ¹³C value of which is within the range of −34‰to −24‰.

The present invention further reveals a liquid polyisoprene polymerwhich is comprised of repeat units that are derived from isoprenemonomer, wherein the liquid polyisoprene polymer has a weight averagemolecular weight which is within the range of 5,000 to 100,000, andwherein the liquid polyisoprene polymer has δ¹³C value of which iswithin the range of −34‰ to −24‰.

The subject invention also disloses a method for verifying that apolyisoprene homopolymer is from a sustainable renewable non-petroleumderived source which comprises: (I) determining the δ¹³C value of thepolyisoprene homopolymer; (II) if the polyisoprene homopolymer has aδ¹³C value within the range of −34‰ to −30‰ or within the range of−28.5‰ to −24‰ additionally analyzing the polyisoprene homopolymer todetermine (1) its cis-microstructure content, (2) its 3,4-microstructurecontent, (3) its 1,2-microstructure content, (4) its a weight averagemolecular weight, or (5) the presence or absence of residual proteins,soaps, lipids, resins, or sugars indicative of natural rubber; and (III)verifying that the polyisoprene homopolymer is from a sustainablerenewable non-petroleum derived source if it has (i) a δ¹³C value ofgreater than −22‰, (ii) a δ¹³C value which is within the range of −30‰to −28.5‰, or (iii) a δ¹³C value within the range of −34‰ to −30‰ orwithin the range of −28.5‰ to −24‰ and if it (a) has acis-microstructure content of less than 100%, (b) contains3,4-microstructure, (c) contains 1,2-microstructure, (d) has a weightaverage molecular weight of less than 100,000, or (e) is free ofresidual proteins, soaps, lipids, resins, or sugars indicative ofnatural rubber.

The present invention further reveals a method for verifying that acopolymer having repeat units that are derived from isoprene containsisoprene that is from a sustainable renewable non-petroleum derivedsource, said method comprising: (I) determining the δ¹³C value of atleast one polyisoprene block in the copolymer; and (II) verifying thatthe isoprene in the copolymer is from a sustainable renewablenon-petroleum derived source if the polyisoprene block has (i) a δ¹³Cvalue of greater than −22‰, or (ii) a δ¹³C value which is within therange of −34‰ to −28.5.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is the nucleotide sequence of a kudzu isoprene synthase genecodon-optimized for expression in E. coli (SEQ ID NO:1). The atg startcodon is in italics, the stop codon is in bold and the added PstI siteis underlined.

FIG. 2 is a map of pTrcKudzu.

FIG. 3 is the nucleotide sequence of pTrcKudzu (SEQ ID NO:2). The RBS isunderlined, the kudzu isoprene synthase start codon is in bold capitolletters and the stop codon is in bold, capitol, italics letters. Thevector backbone is pTrcHis2B.

FIG. 4 is a map of pETNHisKudzu.

FIG. 5 is the nucleotide sequence of pETNHisKudzu (SEQ ID NO:5).

FIG. 6 is a map of pCL-lac-Kudzu.

FIG. 7 is the nucleotide sequence of pCL-lac-Kudzu (SEQ ID NO:7).

FIG. 8A is a graph showing the production of isoprene in E. coli BL21cells with no vector.

FIG. 8B is a graph showing the production of isoprene in E. coli BL21cells with pCL-lac-Kudzu

FIG. 8C is a graph showing the production of isoprene in E. coli BL21cells with pTrcKudzu.

FIG. 8D is a graph showing the production of isoprene in E. coli BL21cells with pETN-HisKudzu.

FIG. 9A is a graph showing OD over time of fermentation of E. coliBL21/pTrcKudzu in a 14 liter fed batch fermentation.

FIG. 9B is a graph showing isoprene production over time of fermentationof E. coli BL21/pTrcKudzu in a 14 liter fed batch fermentation.

FIG. 10A is a graph showing the production of isoprene in Panteoacitrea. Control cells without recombinant kudzu isoprene synthase. Greydiamonds represent isoprene synthesis, black squares represent OD600.

FIG. 10B is a graph showing the production of isoprene in Panteoa citreaexpressing pCL-lac Kudzu. Grey diamonds represent isoprene synthesis,black squares represent OD600.

FIG. 10C is a graph showing the production of isoprene in Panteoa citreaexpressing pTrcKudzu. Grey diamonds represent isoprene synthesis, blacksquares represent OD600.

FIG. 11 is a graph showing the production of isoprene in Bacillussubtilis expressing recombinant isoprene synthase. BG3594comK is a B.subtilis strain without plasmid (native isoprene production).CF443-BG3594comK is a B. subtilis strain with pBSKudzu (recombinantisoprene production). IS on the y-axis indicates isoprene.

FIG. 12 is the nucleotide sequence of pBS Kudzu #2 (SEQ ID NO:57).

FIG. 13 is the nucleotide sequence of kudzu isoprene synthasecodon-optimized for expression in Yarrowia (SEQ ID NO:8).

FIG. 14 is a map of pTrex3g comprising a kudzu isoprene synthase genecodon-optimized for expression in Yarrowia.

FIG. 15 is the nucleotide sequence of vector pSPZ1(MAP29Spb) (SEQ IDNO:11).

FIG. 16 is the nucleotide sequence of the synthetic kudzu (Puerariamontana) isoprene gene codon-optimized for expression in Yarrowia (SEQID NO:12).

FIG. 17 is the nucleotide sequence of the synthetic hybrid poplar(Populus alba×Populus tremula) isoprene synthase gene (SEQ ID NO:13).The ATG start codon is in bold and the stop codon is underlined.

FIG. 18A1 and FIG. 18A2 show a schematic outlining construction ofvectors pYLA 1, pYL1 and pYL2. In FIG. 18A1, YURA51 represents SEQ IDNO:79; YURA3 represents SEQ ID NO:77; Y18S5 represents SEQ ID NO:76; andY18S3 represents SEQ ID NO:75. In FIG. 18A2, XPRT5 represents SEQ IDNO:74; and XPRT3 represents SEQ ID NO:73.

FIG. 18B shows a schematic outlining construction of the vectorpYLA(POP1). In FIG. 18B, XPR5 represents SEQ ID NO:72; and XPR3represents SEQ ID NO:71).

FIG. 18C shows a schematic outlining construction of the vectorpYLA(KZ1)

FIG. 18D shows a schematic outlining construction of the vectorpYLI(KZ1). In FIG. 18D, ICL15 represents SEQ ID NO:70; and ICL13represents SEQ ID NO:69.

FIG. 18E shows a schematic outlining construction of the vectorpYLI(MAP29)

FIG. 18F shows a schematic outlining construction of the vectorpYLA(MAP29)

FIG. 19 shows the MVA and DXP metabolic pathways for isoprene (based onF. Bouvier et al., Progress in Lipid Res. 44: 357-429, 2005). Thefollowing description includes alternative names for each polypeptide inthe pathways and a reference that discloses an assay for measuring theactivity of the indicated polypeptide (each of these references are eachhereby incorporated by reference in their entireties, particularly withrespect to assays for polypeptide activity for polypeptides in the MVAand DXP pathways). Mevalonate Pathway: AACT; Acetyl-CoAacetyltransferase, MvaE, EC 2.3.1.9. Assay: J. Bacteriol., 184:2116-2122, 2002; HMGS; Hydroxymethylglutaryl-CoA synthase, MvaS, EC2.3.3.10. Assay: J. Bacteriol., 184: 4065-4070, 2002; HMGR;3-Hydroxy-3-methylglutaryl-CoA reductase, MvaE, EC 1.1.1.34. Assay: J.Bacteriol., 184: 2116-2122, 2002; MVK; Mevalonate kinase, ERG12, EC2.7.1.36. Assay: Curr Genet. 19:9-14, 1991. PMK; Phosphomevalonatekinase, ERGS, EC 2.7.4.2, Assay: Mol Cell Biol., 11:620-631, 1991;DPMDC; Diphosphomevalonate decarboxylase, MVD1, EC 4.1.1.33. Assay:Biochemistry, 33:13355-13362, 1994; IDI; Isopentenyl-diphosphatedelta-isomerase, IDI1, EC 5.3.3.2. Assay: J. Biol. Chem.264:19169-19175, 1989. DXP Pathway: DXS; 1-Deoxyxylulose-5-phosphatesynthase, dxs, EC 2.2.1.7. Assay: PNAS, 94:12857-62, 1997; DXR;1-Deoxy-D-xylulose 5-phosphate reductoisomerase, dxr, EC 2.2.1.7. Assay:Eur. J. Biochem. 269:4446-4457, 2002; MCT;4-Diphosphocytidyl-2C-methyl-D-erythritol synthase, IspD, EC 2.7.7.60.Assay: PNAS, 97: 6451-6456, 2000; CMK;4-Diphosphocytidyl-2-C-methyl-D-erythritol kinase, IspE, EC 2.7.1.148.Assay: PNAS, 97:1062-1067, 2000; MCS; 2C-Methyl-D-erythritol2,4-cyclodiphosphate synthase, IspF, EC 4.6.1.12. Assay: PNAS,96:11758-11763, 1999; HDS; 1-Hydroxy-2-methyl-2-(E)-butenyl4-diphosphate synthase, ispG, EC 1.17.4.3. Assay: J. Org. Chem.,70:9168-9174, 2005; HDR; 1-Hydroxy-2-methyl-2-(E)-butenyl 4-diphosphatereductase, IspH, EC 1.17.1.2. Assay: JACS, 126:12847-12855, 2004.

FIG. 20 shows graphs representing results of the GC-MS analysis ofisoprene production by recombinant Y. lipolytica strains without (left)or with (right) a kudzu isoprene synthase gene. The arrows indicate theelution time of the authentic isoprene standard.

FIG. 21 is a map of pTrcKudzu yIDI DXS Kan.

FIG. 22 is the nucleotide sequence of pTrcKudzu yIDI DXS Kan (SEQ IDNO:20).

FIG. 23A is a graph showing production of isoprene from glucose inBL21/pTrcKudzukan. Time 0 is the time of induction with IPTG (400 μmol).The x-axis is time after induction; the y-axis is OD600 and the y2-axisis total productivity of isoprene (μg/L headspace or specificproductivity (μg/L headspace/OD). Diamonds represent OD600, circlesrepresent total isoprene productivity (μg/L) and squares representspecific productivity of isoprene (μg/L/OD).

FIG. 23B is a graph showing production of isoprene from glucose inBL21/pTrcKudzu yIDI kan. Time 0 is the time of induction with IPTG (400μmol). The x-axis is time after induction; the y-axis is OD600 and they2-axis is total productivity of isoprene (μg/L headspace or specificproductivity (μg/L headspace/OD). Diamonds represent OD600, circlesrepresent total isoprene productivity (μg/L) and squares representspecific productivity of isoprene (μg/L/OD).

FIG. 23C is a graph showing production of isoprene from glucose inBL21/pTrcKudzu DXS kan. Time 0 is the time of induction with IPTG (400μmol). The x-axis is time after induction; the y-axis is OD600 and they2-axis is total productivity of isoprene (μg/L headspace or specificproductivity (μg/L headspace/OD). Diamonds represent OD600, circlesrepresent total isoprene productivity (μg/L) and squares representspecific productivity of isoprene (μg/L/OD).

FIG. 23D is a graph showing production of isoprene from glucose inBL21/pTrcKudzu yIDI DXS kan. Time 0 is the time of induction with IPTG(400 μmol). The x-axis is time after induction; the y-axis is OD600 andthe y2-axis is total productivity of isoprene (μg/L headspace orspecific productivity (μg/L headspace/OD). Diamonds represent OD600,circles represent total isoprene productivity (μg/L) and squaresrepresent specific productivity of isoprene (μg/L/OD).

FIG. 23E is a graph showing production of isoprene from glucose inBL21/pCL PtrcKudzu. Time 0 is the time of induction with IPTG (400μmol). The x-axis is time after induction; the y-axis is OD600 and they2-axis is total productivity of isoprene (μg/L headspace or specificproductivity (μg/L headspace/OD). Diamonds represent OD600, circlesrepresent total isoprene productivity (μg/L) and squares representspecific productivity of isoprene (μg/L/OD).

FIG. 23F is a graph showing production of isoprene from glucose inBL21/pCL PtrcKudzu yIDI. Time 0 is the time of induction with IPTG (400μmol). The x-axis is time after induction; the y-axis is OD600 and they2-axis is total productivity of isoprene (μg/L headspace or specificproductivity (μg/L headspace/OD). Diamonds represent OD600, circlesrepresent total isoprene productivity (μg/L) and squares representspecific productivity of isoprene (μg/L/OD).

FIG. 23G is a graph showing production of isoprene from glucose inBL21/pCL PtrcKudzu DXS. Time 0 is the time of induction with IPTG (400μmol). The x-axis is time after induction; the y-axis is OD600 and they2-axis is total productivity of isoprene (μg/L headspace or specificproductivity (μg/L headspace/OD). Diamonds represent OD600, circlesrepresent total isoprene productivity (μg/L) and squares representspecific productivity of isoprene (μg/UOD).

FIG. 23H is a graph showing production of isoprene from glucose inBL21/pTrcKudzuIDIDXSkan. The arrow indicates the time of induction withIPTG (400 μmol). The x-axis is time after induction; the y-axis is OD600and the y2-axis is total productivity of isoprene (μg/L headspace orspecific productivity (μg/L headspace/OD). Black diamonds representOD600, black triangles represent isoprene productivity (μg/L) and whitesquares represent specific productivity of isoprene (μg/L/OD).

FIG. 24 is a map of pTrcKKDyIkIS kan.

FIG. 25 is a nucleotide sequence of pTrcKKDyIkIS kan (SEQ ID NO:33).

FIG. 26 is a map of pCL PtrcUpperPathway.

FIG. 27 is a nucleotide sequence of pCL PtrcUpperPathway (SEQ ID NO:46).

FIG. 28 shows a map of the cassette containing the lower MVA pathway andyeast idi for integration into the B. subtilis chromosome at the nprElocus. nprE upstream/downstream indicates 1 kb each of sequence from thenprE locus for integration. aprE promoter (alkaline serine proteasepromoter) indicates the promoter (−35, −10, +1 transcription start site,RBS) of the aprE gene. MVK1 indicates the yeast mevalonate kinase gene.RBS-PMK indicates the yeast phosphomevalonte kinase gene with a BacillusRBS upstream of the start site. RBS-MPD indicates the yeastdiphosphomevalonate decarboxylase gene with a Bacillus RBS upstream ofthe start site. RBS-IDI indicates the yeast idi gene with a Bacillus RBSupstream of the start site. Terminator indicates the terminator alkalineserine protease transcription terminator from B. amyliquefaciens. SpecRindicates the spectinomycin resistance marker. “nprE upstream repeat foramp.” indicates a direct repeat of the upstream region used foramplification.

FIG. 29 is a nucleotide sequence of cassette containing the lower MVApathway and yeast idi for integration into the B. subtilis chromosome atthe nprE locus (SEQ ID NO:47).

FIG. 30 is a map of p9796-poplar.

FIG. 31 is a nucleotide sequence of p9796-poplar (SEQ ID NO:48).

FIG. 32 is a map of pTrcPoplar.

FIG. 33 is a nucleotide sequence of pTrcPoplar (SEQ ID NO:49).

FIG. 34 is a map of pTrcKudzu yIDI Kan.

FIG. 35 is a nucleotide sequence of pTrcKudzu yIDI Kan (SEQ ID NO:50).

FIG. 36 is a map of pTrcKudzuDXS Kan.

FIG. 37 is a nucleotide sequence of pTrcKudzuDXS Kan (SEQ ID NO:51).

FIG. 38 is a map of pCL PtrcKudzu.

FIG. 39 is a nucleotide sequence of pCL PtrcKudzu (SEQ ID NO:52).

FIG. 40 is a map of pCL PtrcKudzu A3.

FIG. 41 is a nucleotide sequence of pCL PtrcKudzu A3 (SEQ ID NO:53).

FIG. 42 is a map of pCL PtrcKudzu yIDI.

FIG. 43 is a nucleotide sequence of pCL PtrcKudzu yIDI (SEQ ID NO:54).

FIG. 44 is a map of pCL PtrcKudzu DXS.

FIG. 45 is a nucleotide sequence of pCL PtrcKudzu DXS (SEQ ID NO:55).

FIG. 46 shows graphs representing isoprene production from biomassfeedstocks. Panel A shows isoprene production from corn stover, Panel Bshows isoprene production from bagasse, Panel C shows isopreneproduction from softwood pulp, Panel D shows isoprene production fromglucose, and Panel E shows isoprene production from cells with noadditional feedstock. Grey squares represent OD600 measurements of thecultures at the indicated times post-inoculation and black trianglesrepresent isoprene production at the indicated times post-inoculation.

FIG. 47A shows a graph representing isoprene production by BL21 (XDE3)pTrcKudzu yIDI DXS (kan) in a culture with no glucose added. Squaresrepresent OD600, and triangles represent isoprene produced (μg/ml).

FIG. 47B shows a graph representing isoprene production from 1% glucosefeedstock invert sugar by BL21 (XDE3) pTrcKudzu yIDI DXS (kan). Squaresrepresent OD600, and triangles represent isoprene produced (μg/ml).

FIG. 47C shows a graph representing isoprene production from 1% invertsugar feedstock by BL21 (λDE3) pTrcKudzu yLDI DXS (kan). Squaresrepresent OD600, and triangles represent isoprene produced (μg/ml).

FIG. 47D shows a graph representing isoprene production from 1% AFEXcorn stover feedstock by BL21 (λDE3) pTrcKudzu yIDI DXS (kan). Squaresrepresent OD600, and triangles represent isoprene produced (μg/ml).

FIG. 48 shows graphs demonstrating the effect of yeast extract ofisoprene production. Panel A shows the time course of optical densitywithin fermentors fed with varying amounts of yeast extract. Panel Bshows the time course of isoprene titer within fermentors fed withvarying amounts of yeast extract. The titer is defined as the amount ofisoprene produced per liter of fermentation broth. Panel C shows theeffect of yeast extract on isoprene production in E. coli grown infed-batch culture.

FIG. 49 shows graphs demonstrating isoprene production from a 500 Lbioreactor with E. coli cells containing the pTrcKudzu+yIDI+DXS plasmid.Panel A shows the time course of optical density within the 500-Lbioreactor fed with glucose and yeast extract. Panel B shows the timecourse of isoprene titer within the 500-L bioreactor fed with glucoseand yeast extract. The titer is defined as the amount of isopreneproduced per liter of fermentation broth. Panel C shows the time courseof total isoprene produced from the 500-L bioreactor fed with glucoseand yeast extract.

FIG. 50 is a map of pJMupperpathway2.

FIG. 51 is the nucleotide sequence of pJMupperpathway2 (SEQ ID NO:56).

FIG. 52 is a map of pBS Kudzu #2.

FIG. 53A is a graph showing growth during fermentation time of Bacillusexpressing recombinant kudzu isoprene synthase in 14 liter fed batchfermentation. Black diamonds represent a control strain (BG3594comK)without recombinant isoprene synthase (native isoprene production) andgrey triangles represent Bacillus with pBSKudzu (recombinant isopreneproduction).

FIG. 53B is a graph showing isoprene production during fermentation timeof Bacillus expressing recombinant kudzu isoprene synthase in 14 literfed batch fermentation. Black diamonds represent a control strain(BG3594comK) without recombinant isoprene synthase (native isopreneproduction) and grey triangles represent Bacillus with pBSKudzu(recombinant isoprene production).

FIG. 54 is a map of plasmid pET24 P. alba HGS.

FIGS. 55A and 55B are the nucleotide sequence of plasmid pET24 P. albaHGS (SEQ ID NO:87).

FIG. 56 is a schematic diagram showing restriction sites used forendonuclease digestion to construct plasmid EWL230 and compatiblecohesive ends between BspHI and NcoI sites.

FIG. 57 is a map of plasmid EWL230.

FIGS. 58A and 58B are the nucleotide sequence of plasmid EWL230 (SEQ IDNO:88).

FIG. 59 is a schematic diagram showing restriction sites used forendonuclease digestion to construct plasmid EWL244 and compatiblecohesive ends between NsiI and PstI sites.

FIG. 60 is a map of EWL244.

FIGS. 61A and 61B are the nucleotide sequence of plasmid EWL244 (SEQ IDNO:89).

FIG. 62 is a map of plasmids MCM484-487.

FIGS. 63A-63C are the nucleotide sequence of plasmid MCM484 (SEQ IDNO:90).

FIGS. 64A-64C are the nucleotide sequence of plasmid MCM485 (SEQ IDNO:91).

FIGS. 65A-65C are the nucleotide sequence of plasmid MCM486 (SEQ IDNO:92).

FIGS. 66A-66C are the nucleotide sequence of plasmid MCM487 (SEQ IDNO:93).

FIGS. 67A-67D are graphs of isoprene production by E. coli strain(EWL256) expressing genes from the MVA pathway and grown in fed-batchculture at the 15-L scale without yeast extract feeding. FIG. 67A showsthe time course of optical density within the 15-L bioreactor fed withglucose. FIG. 67B shows the time course of isoprene titer within the15-L bioreactor fed with glucose. The titer is defined as the amount ofisoprene produced per liter of fermentation broth. FIG. 67C shows thetime course of total isoprene produced from the 15-L bioreactor fed withglucose. FIG. 67D shows the total carbon dioxide evolution rate (TCER),or metabolic activity profile, within the 15-L bioreactor fed withglucose.

FIGS. 68A-68E are graphs of isoprene production by E. coli strain(EWL256) expressing genes from the MVA pathway and grown in fed-batchculture at the 15-L scale with yeast extract feeding. FIG. 68A shows thetime course of optical density within the 15-L bioreactor fed withglucose. FIG. 68B shows the time course of isoprene titer within the15-L bioreactor fed with glucose. The titer is defined as the amount ofisoprene produced per liter of fermentation broth. FIG. 68C shows thetime course of total isoprene produced from the 15-L bioreactor fed withglucose. FIG. 68D shows the volumetric productivity within the 15-Lbioreactor fed with glucose. An average value of 1.1 g/L/hr wasmaintained for a 40-hour period (23-63 hours) with yeast extractfeeding. FIG. 68E shows the carbon dioxide evolution rate (CER), ormetabolic activity profile, within the 15-L bioreactor fed with glucose.

FIGS. 69A-69D shows production of isoprene from different carbon sourcesvia the MVA (pathway). FIG. 69A shows growth of E. coli EWL256, whichcontains both the MVA pathway and isoprene synthase, on either glucose,biomass hydrolysate, glycerol, or acetate as the only carbon source. Thedifferent carbon sources were added to a concentration of 1% in themedia. A negative control with no added carbon source was included.Growth was measured as optical density at 600 nM. FIG. 69B showsspecific productivity of isoprene from E. coli EWL256 containing boththe MVA pathway and isoprene synthase when grown on either glucose,biomass hydrolysate, glycerol, or acetate as only carbon source. Thedifferent carbon sources were added to a concentration of 1% in themedia. A negative control with no added carbon source was included.Samples were taken 190 minutes, 255 minutes and 317 minutes afterinoculation and isoprene produced by the bacteria was measured usingGC-MS. FIG. 69C shows growth of E. coli EWL256 on either glucose orxylose as the only carbon source. The different carbon sources wereadded to a concentration of 1% in the media. A negative control with noadded carbon source was included. Growth was measured as optical densityat 600 nM. FIG. 69D shows specific productivity of isoprene from E. coliEWL256 when grown on either glucose or xylose as only carbon source. Thecarbon sources were added to a concentration of 1% in the media. Anegative control with no added carbon source was included. Samples weretaken 260 minutes, 322 minutes and 383 minutes after inoculation andisoprene produced by the bacteria was measured using GC-MS.

FIGS. 70A and 70B show the production of isoprene by E. coli strainsfrom glucose and from fatty acid, respectively. For FIG. 70A, elevencolonies from the transformation of WW4 with pMCM118, the plasmidbearing the lower mevalonic acid pathway, were picked to verify thepresence of the lower pathway. Cell from the colonies were cultured inTM3 medium containing 0.1% yeast extract and 2% glucose. Aliquots ofinduced culture were assayed for isoprene production after 4 hours ofinduction. All colonies showed the production of isoprene. The inducerIPTG had a strong growth inhibitory effect as was evident from the 3 to4.6-fold reduced cell density in going from 50 to 900 uM concentrationof the inducer (data not shown). The graph shows that higher induction,yields a higher specific titer of isoprene. For FIG. 70B, the productionculture was inoculated from a washed overnight culture at 1 to 10dilution. The culture was grown for several hours and induced with 50 uMIPTG. The left bar shows isoprene assay results four hours afterinduction followed by a one hour isoprene accumulation assay. The middlebar shows the one hour normalized value for the same culture with thesame induction period but analyzed by a 12 hour isoprene accumulationassay. The right bar shows the value for a one hour isopreneaccumulation assay of the culture that was induced for 13 hours.

FIG. 71 is a map of the E. coli-Streptomyces shuttle vector pUWL201PW(6400 bp) used for cloning isoprene synthase from Kudzu. Tsr,thiostrepton resistance gene. Picture is taken from Doumith et al., Mol.Gen. Genet. 264: 477-485, 2000.

FIG. 72 shows isoprene formation by Streptomyces albus wild type strain(“wt”) and strains harboring plasmid pUWL201PW (negative control) orpUWL201_iso (encoding isoprene synthase from Kudzu).

FIG. 73A is a map of the M. mazei archaeal Lower Pathway operon.

FIGS. 73B and 73C are the nucleotide sequence of the M. mazei archaeallower Pathway operon (SEQ ID NO:113).

FIG. 74A is a map of MCM376-MVK from M. mazei archaeal Lowerin pET200D.

FIGS. 74B and 74C are the nucleotide sequence of MCM376-MVK from M.mazei archaeal Lowerin pET200D (SEQ ID NO:114).

FIGS. 75A-75D show growth and specific productivity of isopreneproduction for EWL256 compared to RM11608-2. Growth (OD550) isrepresented by the white diamonds; specific productivity of isoprene isrepresented by the solid bars. The x-axis is time (hours) post-inductionwith either 200 (FIGS. 75A and 75B) or 400 (FIGS. 75C and 75D) uM IPTG.Y-1 axis is productivity of isoprene (ug/L/OD/hr) and Y-2 is arbitraryunits of optical density at a wavelength of 550. These values for theOD550 must be multiplied by 6.66 to obtain the actual OD of the culture.

FIG. 76 is a map of plasmid pBBRCMPGI1.5-pgl.

FIGS. 77A and 77B are the nucleotide sequence of plasmidpBBRCMPGI1.5-pgl (SEQ ID NO:122).

FIGS. 78A-78F are graphs of isoprene production by E. coli strainexpressing M. mazei mevalonate kinase, P. alba isoprene synthase, andpgl (RHM111608-2), and grown in fed-batch culture at the 15-L scale.FIG. 78A shows the time course of optical density within the 15-Lbioreactor fed with glucose. FIG. 78B shows the time course of isoprenetiter within the 15-L bioreactor fed with glucose. The titer is definedas the amount of isoprene produced per liter of fermentation broth.Method for calculating isoprene: cumulative isoprene produced in 59 hrs,g/Fermentor volume at 59 hrs, L [=] g/L broth. FIG. 78C also shows thetime course of isoprene titer within the 15-L bioreactor fed withglucose. Method for calculating isoprene: ∫(Instantaneous isopreneproduction rate, g/L/hr)dt from t=0 to 59 hours [=] g/L broth. FIG. 78Dshows the time course of total isoprene produced from the 15-Lbioreactor fed with glucose. FIG. 78E shows volumetric productivitywithin the 15-L bioreactor fed with glucose. FIG. 78F shows carbondioxide evolution rate (CER), or metabolic activity profile, within the15-L bioreactor fed with glucose.

FIG. 79A is a map of plasmid pJ201:19813.

FIGS. 79B and 79C are the nucleotide sequence of pJ201:19813 (SEQ IDNO:123).

FIG. 80 shows the time course of optical density within the 15-Lbioreactor fed with glucose.

FIG. 81 shows the time course of isoprene titer within the 15-Lbioreactor fed with glucose. The titer is defined as the amount ofisoprene produced per liter of fermentation broth.

FIG. 82 shows the time course of total isoprene produced from the 15-Lbioreactor fed with glucose.

FIG. 83 is a graph illustrating the time course of optical densitywithin the 500-L bioreactor fed with glucose and yeast extract.

FIG. 84 is a graph illustrating the time course of isoprene titer withinthe 500-L bioreactor fed with glucose and yeast extract. The titer isdefined as the amount of isoprene produced per liter of fermentationbroth.

FIG. 85 is a graph illustrating the time course of total isopreneproduced form the 500-L bioreactor fed with glucose and yeast extract.

DETAILED DESCRIPTION OF THE INVENTION

A technique for producing isoprene in a culture of cells that produceisoprene is described in U.S. Provisional Patent Application Ser. No.61/013,574, filed on Dec. 13, 2007, and in U.S. Provisional PatentApplication Ser. No. 61/013,386, filed on Dec. 13, 2007, and in U.S.patent application Ser. No. 12/335,071. The teachings of U.S.Provisional Patent Application Ser. No. 61/013,574 (now published asUnited States Patent Publication No. 2009/0203102A1), U.S. ProvisionalPatent Application Ser. No. 61/013,386, and U.S. patent application Ser.No. 12/335,071 (now published as United States Patent Publication No.2009/0203102A1) are incorporated herein by reference for the purpose ofteaching techniques for producing and recovering isoprene by such aprocess. In any case, U.S. Provisional Patent Application Ser. No.61/013,574, U.S. Provisional Patent Application Ser. No. 61/013,386, andU.S. patent application Ser. No. 12/335,071 teach compositions andmethods for the production of increased amounts of isoprene in cellcultures. In particular, these compositions and methods increase therate of isoprene production and increase the total amount of isoprenethat is produced. For example, cell culture systems that generate4.8×10⁴ nmole/g_(wcm)/hr of isoprene have been produced (Table 1). Theefficiency of these systems is demonstrated by the conversion of ˜23.6molar % yield (10.7 weight % yield) of the carbon that the cells consumefrom a cell culture medium into isoprene (% carbon yield). As shown inthe Examples and Table 2, approximately 60.5 g of isoprene per liter ofbroth was generated. Isoprene was produced at a peak specific rate of1.88×10⁵ nmol/OD/hr (1.88×10⁵ nmole/g_(wcm)/hr). If desired, evengreater amounts of isoprene can be obtained using other conditions, suchas those described herein. In some embodiments, a renewable carbonsource is used for the production of isoprene. The compositions andmethods of the present invention are desirable because they allow highisoprene yield per cell, high carbon yield, high isoprene purity, highproductivity, low energy usage, low production cost and investment, andminimal side reactions. This efficient, large scale, biosyntheticprocess for isoprene production provides an isoprene source forsynthetic isoprene-based rubber and provides a desirable, low-costalternative to using natural rubber.

As discussed further below, the amount of isoprene produced by cells canbe greatly increased by introducing a heterologous nucleic acid encodingan isoprene synthase polypeptide (e.g., a plant isoprene synthasepolypeptide) into the cells. Isoprene synthase polypeptides convertdimethylallyl diphosphate (DMAPP) into isoprene. As shown in theExamples, a heterologous Pueraria Montana (kudzu) or Populus alba(Poplar) isoprene synthase polypeptide was expressed in a variety ofhost cells, such as Escherichia coli, Panteoa citrea, Bacillus subtilis,Yarrowia lipolytica, and Trichoderma reesei. As also shown in theExamples, a heterologous Methanosarcina mazei (M. mazei) mevalonatekinase (MVK) was expressed in host cells such as Escherichia coli toincrease isoprene production. All of these cells produced more isoprenethan the corresponding cells without the heterologous isoprene synthasepolypeptide. As illustrated in Tables 1 and 2, large amounts of isopreneare produced using the methods described herein. For example, B.subtilis cells with a heterologous isoprene synthase nucleic acidproduced approximately 10-fold more isoprene in a 14 liter fermentorthan the corresponding control B. subtilis cells without theheterologous nucleic acid (Table 2). The production of 60.5 g ofisoprene per liter of broth (mg/L, wherein the volume of broth includesboth the volume of the cell medium and the volume of the cells) by E.coli and 30 mg/L by B. subtilis in fermentors indicates that significantamounts of isoprene can be generated (Table 2). If desired, isoprene canbe produced on an even larger scale or other conditions described hereincan be used to further increase the amount of isoprene. The vectorslisted in Tables 1 and 2 and the experimental conditions are describedin further detail below and in the Examples section.

TABLE 1 Exemplary yields of isoprene from a shake flask using the cellcultures and methods of the invention. Isoprene Production in aHeadspace vial* Headspace Specific Rate concentration μg/L_(broth)/hr/ODStrain μg/L_(gas) (nmol/g_(wcm)/hr) E. coli BL21/pTrcKudzu IS 1.40 53.2(781.2)  E. coli BL21/Pcl DXS yidi 7.61 289.1  Kudzu IS (4.25 × 10³) E.coli BL21/MCM127 with 23.0 874.1  kudzu IS and entire MVA (1.28 × 10⁴)pathway E. coli BL21/Pet N- 1.49 56.6 HisKudzu IS (831.1)  Pantoeacitrea/pTrcKudzu 0.66 25.1 IS (368.6)  E. coli w/Poplar IS —  5.6[Miller (2001)] (82.2) Bacillis licheniformis Fall —  4.2 U.S. Pat. No.5,849,970 (61.4) Yarrowia lipolytica with ~0.05 μg/L ~2   kudzu isoprenesynthase (~30)   Trichoderma reesei with ~0.05 μg/L ~2   kudzu isoprenesynthase (~30)   E. coli BL21/ 85.9 3.2 × 10³ pTrcKKD_(y)I_(k)IS withkudzu (4.8 × 10⁴) IS and lower MVA pathway The assay for measuringisoprene production is described in Example I, part II. For this assay,a sample was removed at one or more time points from the shake flask andcultured for 30 minutes. The amount of isoprene produced in this samplewas then measured. The headspace concentration and specific rate ofisoprene production are listed in Table 1 and described further herein.*Normalized to 1 mL of 1 OD₆₀₀, cultured for 1 hour in a sealedheadspace vial with a liquid to headspace volume ratio of 1:19.

TABLE 2 Exemplary yields of isoprene in a fermentor using the cellcultures and methods of the invention. Isoprene Production in FermentorsPeak Headspace Peak Specific rate concentration** Titerμg/L_(broth)/hr/OD Strain (ug/L_(gas)) (mg/L_(broth)) (nmol/g_(wcm)/hr)E. coli BL21/ 52 41.2 37  pTrcKudzu with (543.3) Kudzu IS E. coli 3 3.5 21.4 FM5/pTrcKudzu IS (308.1) E. coli BL21/triple 285 300 240   strain(DXS, yidi, (3.52 × 10³) IS) E. coli FM5/triple 50.8 29 180.8 strain(DXS, yidi, (2.65 × 10³) IS) E. coli/MCM127 1094 250 875   with Kudzu ISand (1.28 × 10⁴) entire MVA pathway Bacillus subtilis 1.5 2.5  0.8wild-type  (11.7) Bacillus pBS Kudzu 16.6 ~30 5 IS (over 100  (73.4)hours) Bacillus Marburg 2.04 0.61  24.5 6051 [Wagner and (359.8) Fall(1999)] Bacillus Marburg 0.7 0.15  6.8 6051 Fall U.S. (100)   Pat. No.5,849,970 E. coli 2.03 × 10⁴ 3.22 × 10⁴  5.9 × 10³ BL21/pCLPtrcUpper(8.66 × 10⁴) Pathway and gil.2KKDyI and pTrcAlba-mMVK E. coli 3.22 × 10⁴6.05 × 10⁴ 1.28 × 10⁴ BL21/pCLPtrcUpper (1.88 × 10⁵) Pathway andgi1.2KKDyI and pTrcAlba-mMVK plus pBBRCMPGI1.5pgl The assay formeasuring isoprene production is described in Example I, part II. Forthis assay, a sample of the off-gas of the fermentor was taken andanalyzed for the amount of isoprene. The peak headspace concentration(which is the highest headspace concentration during the fermentation),titer (which is the cumulative, total amount of isoprene produced perliter of broth), and peak specific rate of isoprene production (which isthe highest specific rate during the fermentation) are listed in Table 2and described further herein. **Normalized to an off-gas flow rate of 1vvm (1 volume off-gas per 1 L_(broth) per minute).

Additionally, isoprene production by cells that contain a heterologousisoprene synthase nucleic acid can be enhanced by increasing the amountof a 1-deoxy-D-xylulose-5-phosphate synthase (DXS) polypeptide and/or anisopentenyl diphosphate isomerase (IDI) polypeptide expressed by thecells. For example, a DXS nucleic acid and/or an IDI nucleic acid can beintroduced into the cells. The DXS nucleic acid may be a heterologousnucleic acid or a duplicate copy of an endogenous nucleic acid.Similarly, the IDI nucleic acid may be a heterologous nucleic acid or aduplicate copy of an endogenous nucleic acid. In some embodiments, theamount of DXS and/or IDI polypeptide is increased by replacing theendogenous DXS and/or IDI promoters or regulatory regions with otherpromoters and/or regulatory regions that result in greater transcriptionof the DXS and/or IDI nucleic acids. In some embodiments, the cellscontain both a heterologous nucleic acid encoding an isoprene synthasepolypeptide (e.g., a plant isoprene synthase nucleic acid) and aduplicate copy of an endogenous nucleic acid encoding an isoprenesynthase polypeptide.

The encoded DXS and IDI polypeptides are part of the DXP pathway for thebiosynthesis of isoprene (FIG. 19A). DXS polypeptides convert pyruvateand D-glyceraldehyde-3-phosphate into 1-deoxy-D-xylulose-5-phosphate.While not intending to be bound by any particular theory, it is believedthat increasing the amount of DXS polypeptide increases the flow ofcarbon through the DXP pathway, leading to greater isoprene production.IDI polypeptides catalyze the interconversion of isopentenyl diphosphate(IPP) and dimethylallyl diphosphate (DMAPP). While not intending to bebound by any particular theory, it is believed that increasing theamount of IDI polypeptide in cells increases the amount of IPP that isconverted into DMAPP, which in turn is converted into isoprene.

For example, fermentation of E. coli cells with a kudzu isoprenesynthase, S. cerevisia IDI, and E. coli DXS nucleic acids was used toproduce isoprene. The levels of isoprene varied from 50 to 300 μg/L overa time period of 15 hours (Example 7, part VII).

As another example, fermentation of E. coli with M. mazei mevalonatekinase (MVK), P. alba isoprene synthase, the upper MVA pathway, and theintegrated lower MVA pathway was used to produce isoprene. The levels ofisoprene varied from 32 to 35.6 g/L over a time period of 67 hours(Example 10, part III).

In yet another example, fermentation of E. coli with M. mazei mevalonatekinase (MVK), P. alba isoprene synthase, pgl over-expression(RHM111608-2), the upper MVA pathway, and the integrated lower MVApathway were used to produce isoprene. The levels of isoprene vary from33.2 g/L to 40.0 g/L over a time period of 40 hours or 48.6 g/L to 60.5g/L over a time period of 59 hours (Example 13, part (ii)).

In some embodiments, the presence of heterologous or extra endogenousisoprene synthase, IDI, and DXS nucleic acids causes cells to grow morereproducibly or remain viable for longer compared to the correspondingcell with only one or two of these heterologous or extra endogenousnucleic acids. For example, cells containing heterologous isoprenesynthase, IDI, and DXS nucleic acids grew better than cells with onlyheterologous isoprene synthase and DXS nucleic acids or with only aheterologous isoprene synthase nucleic acid. Also, heterologous isoprenesynthase, IDI, and DXS nucleic acids were successfully operably linkedto a strong promoter on a high copy plasmid that was maintained by E.coli cells, suggesting that large amounts of these polypeptides could beexpressed in the cells without causing an excessive amount of toxicityto the cells. While not intending to be bound to a particular theory, itis believed that the presence of heterologous or extra endogenousisoprene synthase and IDI nucleic acids may reduce the amount of one ormore potentially toxic intermediates that would otherwise accumulate ifonly a heterologous or extra endogenous DXS nucleic acid was present inthe cells.

In some embodiments, the production of isoprene by cells that contain aheterologous isoprene synthase nucleic acid is augmented by increasingthe amount of a MVA polypeptide expressed by the cells (FIGS. 19A and19B). Exemplary MVA pathways polypeptides include any of the followingpolypeptides: acetyl-CoA acetyltransferase (AA-CoA thiolase)polypeptides, 3-hydroxy-3-methylglutaryl-CoA synthase (HMG-CoA synthase)polypeptides, 3-hydroxy-3-methylglutaryl-CoA reductase (HMG-CoAreductase) polypeptides, mevalonate kinase (MVK) polypeptides,phosphomevalonate kinase (PMK) polypeptides, diphosphomevalontedecarboxylase (MVD) polypeptides, phosphomevalonate decarboxylase (PMDC)polypeptides, isopentenyl phosphate kinase (IPK) polypeptides, IDIpolypeptides, and polypeptides (e.g., fusion polypeptides) having anactivity of two or more MVA pathway polypeptides. For example, one ormore MVA pathway nucleic acids can be introduced into the cells. In someembodiments, the cells contain the upper MVA pathway, which includesAA-CoA thiolase, HMG-CoA synthase, and HMG-CoA reductase nucleic acids.In some embodiments, the cells contain the lower MVA pathway, whichincludes MVK, PMK, MVD, and IDI nucleic acids. In some embodiments, thecells contain the entire MVA pathway, which includes AA-CoA thiolase,HMG-CoA synthase, HMG-CoA reductase, MVK, PMK, MVD, and IDI nucleicacids. In some embodiments, the cells contain an entire MVA pathway thatincludes AA-CoA thiolase, HMG-CoA synthase, HMG-CoA reductase, MVK,PMDC, IPK, and IDI nucleic acids. The MVA pathway nucleic acids may beheterologous nucleic acids or duplicate copies of endogenous nucleicacids. In some embodiments, the amount of one or more MVA pathwaypolypeptides is increased by replacing the endogenous promoters orregulatory regions for the MVA pathway nucleic acids with otherpromoters and/or regulatory regions that result in greater transcriptionof the MVA pathway nucleic acids. In some embodiments, the cells containboth a heterologous nucleic acid encoding an isoprene synthasepolypeptide (e.g., a plant isoprene synthase nucleic acid) and aduplicate copy of an endogenous nucleic acid encoding an isoprenesynthase polypeptide.

For example, E. coli cells containing a nucleic acid encoding a kudzuisoprene synthase polypeptide and nucleic acids encoding Saccharomycescerevisia MVK, PMK, MVD, and IDI polypeptides generated isoprene at arate of 6.67×10⁴ nmol/L_(broth)/OD₆₀₀/hr (see Example 8). Additionally,a 14 liter fermentation of E. coli cells with nucleic acids encodingEnterococcus faecalis AA-CoA thiolase, HMG-CoA synthase, and HMG-CoAreductase polypeptides produced 22 grams of mevalonic acid (anintermediate of the MVA pathway). A shake flask of these cells produced2-4 grams of mevalonic acid per liter. These results indicate thatheterologous MVA pathways nucleic acids are active in E. coli. E. colicells that contain nucleic acids for both the upper MVA pathway and thelower MVA pathway as well as a kudzu isoprene synthase (strain MCM 127)produced significantly more isoprene (874 μg/L_(broth)/hr/OD) comparedto E. coli cells with nucleic acids for only the lower MVA pathway andthe kudzu isoprene synthase (strain MCM 131) (see Table 3 ni isoprenesynthase polypeptide and a nucleic acid encoding M. mazei MVKpolypeptide generated 320.6 g (at a peak specific rate of 9.54×10⁴nmol/L_(broth)/OD₆₀₀/hr (i.e. 9.5×10⁻⁵ mol/L_(broth)/OD₆₀₀/hr)) ofisoprene during a 67 hour fermentation in the absence of yeast extractfeeding or 395.5 g (at a peak specific rate of 8.66×10⁴nmol/L_(broth)/OD₆₀₀/hr during a 68 hour fermentation in the presence ofyeast extract feeding (see Example 10).

In some embodiments, at least a portion of the cells maintain theheterologous isoprene synthase, DXS, IDI, and/or MVA pathway nucleicacid for at least about 5, 10, 20, 50, 75, 100, 200, 300, or more celldivisions in a continuous culture (such as a continuous culture withoutdilution). In some embodiments of any of the aspects of the invention,the nucleic acid comprising the heterologous or duplicate copy of anendogenous isoprene synthase, DXS, IDI, and/or MVA pathway nucleic acidalso comprises a selective marker, such as a kanamycin, ampicillin,carbenicillin, gentamicin, hygromycin, phleomycin, bleomycin, neomycin,or chloramphenicol antibiotic resistance nucleic acid.

As indicated in Example 7, part VI, the amount of isoprene produced canbe further increased by adding yeast extract to the cell culture mediumusing E. coli cells with kudzu isoprene synthase, S. cerevisia IDI, andE. coli DXS nucleic acids to produce isoprene. In particular, the amountof isoprene produced was linearly proportional to the amount of yeastextract in the cell medium for the concentrations tested (FIG. 48C).Additionally, approximately 0.11 grams of isoprene per liter of brothwas produced from a cell medium with yeast extract and glucose (Example7, part VIII). Increasing the amount of yeast extract in the presence ofglucose resulted in more isoprene being produced than increasing theamount of glucose in the presence of yeast extract. Also, increasing theamount of yeast extract allowed the cells to produce a high level ofisoprene for a longer length of time and improved the health of thecells.

Isoprene production was also demonstrated using three types ofhydrolyzed biomass (bagasse, corn stover, and soft wood pulp) as thecarbon source (FIGS. 46A-C and FIGS. 69A and 69B). E. coli cells withkudzu isoprene synthase, S. cerevisia IDI, and E. coli DXS nucleic acidsproduced as much isoprene from these hydrolyzed biomass carbon sourcesas from the equivalent amount of glucose (e.g., 1% glucose, w/v). E.coli cells expressing P. alba isoprene synthase and the MVA pathwayproduced isoprene at a higher initial growth rate from ammonia fiberexpansion (AFEX) pretreated corn stover than from the equivalent amountof glucose. (FIGS. 69A and 69B). If desired, any other biomass carbonsource can be used in the compositions and methods of the invention.Biomass carbon sources are desirable because they are cheaper than manyconventional cell mediums, thereby facilitating the economicalproduction of isoprene.

Additionally, invert sugar was shown to function as a carbon source forthe generation of isoprene (FIG. 47D).

Additionally, xylose, acetate, and glycerol were also shown to functionas a carbon source for the generation of isoprene (FIGS. 69A-69D). Forexample, E. coli cells with P. alba isoprene synthase and the MVApathway grown on acetate as the only carbon source had a specificproductivity of isoprene about twice as high as during growth on glucose(Example 10, Part IV; FIGS. 69A and 69B).

In some embodiments, an oil is included in the cell medium. For example,B. subtilis cells containing a kudzu isoprene synthase nucleic acidproduced isoprene when cultured in a cell medium containing an oil and asource of glucose (Example 4, part III). As another example, E. colifadR atoC mutant cells containing the upper and lower MVA pathway pluskudzu isoprene synthase produced isoprene when cultured in a cell mediumcontaining palm oil and a source of glucose (Example 12, part II). Insome embodiments, more than one oil (such as 2, 3, 4, 5, or more oils)is included in the cell medium. While not intending to be bound to anyparticular theory, it is believed that (i) the oil may increase theamount of carbon in the cells that is available for conversion toisoprene, (ii) the oil may increase the amount of acetyl-CoA in thecells, thereby increasing the carbon flow through the MVA pathway,and/or (ii) the oil may provide extra nutrients to the cells, which isdesirable since a lot of the carbon in the cells is converted toisoprene rather than other products. In some embodiments, cells that arecultured in a cell medium containing oil naturally use the MVA pathwayto produce isoprene or are genetically modified to contain nucleic acidsfor the entire MVA pathway. In some embodiments, the oil is partially orcompletely hydrolyzed before being added to the cell culture medium tofacilitate the use of the oil by the host cells.

Exemplary Polypeptides and Nucleic Acids

Various isoprene synthase, DXS, IDI, and/or MVA pathway polypeptides andnucleic acids can be used in the compositions and methods of theinvention.

As used herein, “polypeptides” includes polypeptides, proteins,peptides, fragments of polypeptides, and fusion polypeptides thatinclude part or all of a first polypeptide (e.g., an isoprene synthase,DXS, IDI, or MVA pathway polypeptide) and part or all of a secondpolypeptide (e.g., a peptide that facilitates purification or detectionof the fusion polypeptide, such as a His-tag). In some embodiments, thefusion polypeptide has an activity of two or more MVA pathwaypolypeptides (such as AA-CoA thiolase and HMG-CoA reductasepolypeptides). In some embodiments, the polypeptide is anaturally-occurring polypeptide (such as the polypeptide encoded by anEnterococcus faecalis mvaE nucleic acid) that has an activity of two ormore MVA pathway polypeptides.

In various embodiments, a polypeptide has at least or about 50, 100,150, 175, 200, 250, 300, 350, 400, or more amino acids. In someembodiments, the polypeptide fragment contains at least or about 25, 50,75, 100, 150, 200, 300, or more contiguous amino acids from afull-length polypeptide and has at least or about 5%, 10%, 20%, 30%,40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100% of an activity of acorresponding full-length polypeptide. In particular embodiments, thepolypeptide includes a segment of or the entire amino acid sequence ofany naturally-occurring isoprene synthase, DXS, IDI, or MVA pathwaypolypeptide. In some embodiments, the polypeptide has one or moremutations compared to the sequence of a wild-type (i.e., a sequenceoccurring in nature) isoprene synthase, DXS, IDI, or MVA pathwaypolypeptide.

In some embodiments, the polypeptide is an isolated polypeptide. As usedherein, an “isolated polypeptide” is not part of a library ofpolypeptides, such as a library of 2, 5, 10, 20, 50 or more differentpolypeptides and is separated from at least one component with which itoccurs in nature. An isolated polypeptide can be obtained, for example,by expression of a recombinant nucleic acid encoding the polypeptide.

In some embodiments, the polypeptide is a heterologous polypeptide. By“heterologous polypeptide” is meant a polypeptide whose amino acidsequence is not identical to that of another polypeptide naturallyexpressed in the same host cell.

As used herein, a “nucleic acid” refers to two or moredeoxyribonucleotides and/or ribonucleotides in either single ordouble-stranded form. In some embodiments, the nucleic acid is arecombinant nucleic acid. By “recombinant nucleic acid” means a nucleicacid of interest that is free of one or more nucleic acids (e.g., genes)which, in the genome occurring in nature of the organism from which thenucleic acid of interest is derived, flank the nucleic acid of interest.The term therefore includes, for example, a recombinant DNA which isincorporated into a vector, into an autonomously replicating plasmid orvirus, or into the genomic DNA of a prokaryote or eukaryote, or whichexists as a separate molecule (e.g., a cDNA, a genomic DNA fragment, ora cDNA fragment produced by PCR or restriction endonuclease digestion)independent of other sequences. In some embodiments, an isoprenesynthase, DXS, IDI, or MVA pathway nucleic acid is operably linked toanother nucleic acid encoding all or a portion of another polypeptidesuch that the recombinant nucleic acid encodes a fusion polypeptide thatincludes an isoprene synthase, DXS, IDI, or MVA pathway polypeptide andall or part of another polypeptide (e.g., a peptide that facilitatespurification or detection of the fusion polypeptide, such as a His-tag).In some embodiments, part or all of a recombinant nucleic acid ischemically synthesized.

In some embodiments, the nucleic acid is a heterologous nucleic acid. By“heterologous nucleic acid” is meant a nucleic acid whose nucleic acidsequence is not identical to that of another nucleic acid naturallyfound in the same host cell. In particular embodiments, the nucleic acidincludes a segment of or the entire nucleic acid sequence of anynaturally-occurring isoprene synthase, DXS, IDI, or MVA pathway nucleicacid. In some embodiments, the nucleic acid includes at least or about50, 100, 150, 200, 300, 400, 500, 600, 700, 800, or more contiguousnucleotides from a naturally-occurring isoprene synthase nucleic acidDXS, IDI, or MVA pathway nucleic acid. In some embodiments, the nucleicacid has one or more mutations compared to the sequence of a wild-type(i.e., a sequence occurring in nature) isoprene synthase, DXS, IDI, orMVA pathway nucleic acid. In some embodiments, the nucleic acid has oneor more mutations (e.g., a silent mutation) that increase thetranscription or translation of isoprene synthase, DXS, BM, or MVApathway nucleic acid. In some embodiments, the nucleic acid is adegenerate variant of any nucleic acid encoding an isoprene synthase,DXS, IDI, or MVA pathway polypeptide.

“Codon degeneracy” refers to divergence in the genetic code permittingvariation of the nucleotide sequence without affecting the amino acidsequence of an encoded polypeptide. The skilled artisan is well aware ofthe “codon-bias” exhibited by a specific host cell in usage ofnucleotide codons to specify a given amino acid. Therefore, whensynthesizing a nucleic acid for improved expression in a host cell, itis desirable in some embodiments to design the nucleic acid such thatits frequency of codon usage approaches the frequency of preferred codonusage of the host cell.

The accession numbers of exemplary isoprene synthase, DXS, IDI, and/orMVA pathway polypeptides and nucleic acids are listed in Appendix 1 (theaccession numbers of Appendix 1 and their corresponding sequences areherein incorporated by reference in their entireties, particularly withrespect to the amino acid and nucleic acid sequences of isoprenesynthase, DXS, IDI, and/or MVA pathway polypeptides and nucleic acids).The Kegg database also contains the amino acid and nucleic acidsequences of numerous exemplary isoprene synthase, DXS, IDI, and/or MVApathway polypeptides and nucleic acids (see, for example, the world-wideweb at “genome.jp/kegg/pathway/map/map00100.html” and the sequencestherein, which are each hereby incorporated by reference in theirentireties, particularly with respect to the amino acid and nucleic acidsequences of isoprene synthase, DXS, IDI, and/or MVA pathwaypolypeptides and nucleic acids). In some embodiments, one or more of theisoprene synthase, DXS, IDI, and/or MVA pathway polypeptides and/ornucleic acids have a sequence identical to a sequence publicly availableon Dec. 12, 2007 or Dec. 11, 2008, such as any of the sequences thatcorrespond to any of the accession numbers in Appendix 1 or any of thesequences present in the Kegg database. Additional exemplary isoprenesynthase, DXS, IDI, and/or MVA pathway polypeptides and nucleic acidsare described further below.

Exemplary Isoprene Synthase Polypeptides and Nucleic Acids

As noted above, isoprene synthase polypeptides convert dimethylallyldiphosphate (DMAPP) into isoprene. Exemplary isoprene synthasepolypeptides include polypeptides, fragments of polypeptides, peptides,and fusions polypeptides that have at least one activity of an isoprenesynthase polypeptide. Standard methods can be used to determine whethera polypeptide has isoprene synthase polypeptide activity by measuringthe ability of the polypeptide to convert DMAPP into isoprene in vitro,in a cell extract, or in vivo. In an exemplary assay, cell extracts areprepared by growing a strain (e.g., the E. coli/pTrcKudzu straindescribed herein) in the shake flask method as described in Example 1.After induction is complete, approximately 10 mLs of cells are pelletedby centrifugation at 7000×g for 10 minutes and resuspended in 5 ml ofPEB without glycerol. The cells are lysed using a French Pressure cellusing standard procedures. Alternatively the cells are treated withlysozyme (Ready-Lyse lysozyme solution; EpiCentre) after a freeze/thawat −80 C.

Isoprene synthase polypeptide activity in the cell extract can bemeasured, for example, as described in Silver et al., J. Biol. Chem.270:13010-13016, 1995 and references therein, which are each herebyincorporated by reference in their entireties, particularly with respectto assays for isoprene synthase polypeptide activity. DMAPP (Sigma) isevaporated to dryness under a stream of nitrogen and rehydrated to aconcentration of 100 mM in 100 mM potassium phosphate buffer pH 8.2 andstored at −20° C. To perform the assay, a solution of 5 μl of 1M MgCl₂,1 mM (250 μg/ml) DMAPP, 65 μl of Plant Extract Buffer (PEB) (50 mMTris-HCl, pH 8.0, 20 mM MgCl₂, 5% glycerol, and 2 mM DTT) is added to 25μl of cell extract in a 20 ml Headspace vial with a metal screw cap andteflon coated silicon septum (Agilent Technologies) and cultured at 37°C. for 15 minutes with shaking. The reaction is quenched by adding 200μl of 250 mM EDTA and quantified by GC/MS as described in Example 1,part II.

Exemplary isoprene synthase nucleic acids include nucleic acids thatencode a polypeptide, fragment of a polypeptide, peptide, or fusionpolypeptide that has at least one activity of an isoprene synthasepolypeptide. Exemplary isoprene synthase polypeptides and nucleic acidsinclude naturally-occurring polypeptides and nucleic acids from any ofthe source organisms described herein as well as mutant polypeptides andnucleic acids derived from any of the source organisms described herein.

In some embodiments, the isoprene synthase polypeptide or nucleic acidis from the family Fabaceae, such as the Faboideae subfamily. In someembodiments, the isoprene synthase polypeptide or nucleic acid is apolypeptide or nucleic acid from Pueraria montana (kudzu) (Sharkey etal., Plant Physiology 137: 700-712, 2005), Pueraria lobata, poplar (suchas Populus alba, Populus nigra, Populus trichocarpa, Populusalba×tremula (CAC35696), or Populus alba) (Sasaki et al., FEBS Letters579(11): 2514-2518, 2005; Miller et al., Planta 213: 483-487, 2001),aspen (such as Populus tremuloides) (Silver et al., JBC 270(22):13010-1316, 1995), or English Oak (Quercus robur) (Zimmer et al., WO98/02550), which are each hereby incorporated by reference in theirentireties, particularly with respect to isoprene synthase nucleic acidsand the expression of isoprene synthase polypeptides. Suitable isoprenesynthases include, but are not limited to, those identified by GenbankAccession Nos. AY341431, AY316691, AY279379, AJ457070, and AY182241,which are each hereby incorporated by reference in their entireties,particularly with respect to sequences of isoprene synthase nucleicacids and polypeptides. In some embodiments, the isoprene synthasepolypeptide or nucleic acid is not a naturally-occurring polypeptide ornucleic acid from Quercus robur (i.e., the isoprene synthase polypeptideor nucleic acid is an isoprene synthase polypeptide or nucleic acidother than a naturally-occurring polypeptide or nucleic acid fromQuercus robur). In some embodiments, the isoprene synthase nucleic acidor polypeptide is a naturally-occurring polypeptide or nucleic acid frompoplar. In some embodiments, the isoprene synthase nucleic acid orpolypeptide is not a naturally-occurring polypeptide or nucleic acidfrom poplar.

Exemplary DXS Polypeptides and Nucleic Acids

As noted above, 1-deoxy-D-xylulose-5-phosphate synthase (DXS)polypeptides convert pyruvate and D-glyceraldehyde-3-phosphate into1-deoxy-D-xylulose-5-phosphate. Exemplary DXS polypeptides includepolypeptides, fragments of polypeptides, peptides, and fusionspolypeptides that have at least one activity of a DXS polypeptide.Standard methods (such as those described herein) can be used todetermine whether a polypeptide has DXS polypeptide activity bymeasuring the ability of the polypeptide to convert pyruvate andD-glyceraldehyde-3-phosphate into 1-deoxy-D-xylulose-5-phosphate invitro, in a cell extract, or in vivo. Exemplary DXS nucleic acidsinclude nucleic acids that encode a polypeptide, fragment of apolypeptide, peptide, or fusion polypeptide that has at least oneactivity of a DXS polypeptide. Exemplary DXS polypeptides and nucleicacids include naturally-occurring polypeptides and nucleic acids fromany of the source organisms described herein as well as mutantpolypeptides and nucleic acids derived from any of the source organismsdescribed herein.

Exemplary IDI Polypeptides and Nucleic Acids

Isopentenyl diphosphate isomerase polypeptides (isopentenyl-diphosphatedelta-isomerase or IDI) catalyses the interconversion of isopentenyldiphosphate (IPP) and dimethylallyl diphosphate (DMAPP) (e.g.,converting IPP into DMAPP and/or converting DMAPP into IPP). ExemplaryIDI polypeptides include polypeptides, fragments of polypeptides,peptides, and fusions polypeptides that have at least one activity of anIDI polypeptide. Standard methods (such as those described herein) canbe used to determine whether a polypeptide has IDI polypeptide activityby measuring the ability of the polypeptide to interconvert IPP andDMAPP in vitro, in a cell extract, or in vivo. Exemplary IDI nucleicacids include nucleic acids that encode a polypeptide, fragment of apolypeptide, peptide, or fusion polypeptide that has at least oneactivity of an IDI polypeptide. Exemplary IDI polypeptides and nucleicacids include naturally-occurring polypeptides and nucleic acids fromany of the source organisms described herein as well as mutantpolypeptides and nucleic acids derived from any of the source organismsdescribed herein.

Exemplary MVA Pathway Polypeptides and Nucleic Acids

Exemplary MVA pathway polypeptides include acetyl-CoA acetyltransferase(AA-CoA thiolase) polypeptides, 3-hydroxy-3-methylglutaryl-CoA synthase(HMG-CoA synthase) polypeptides, 3-hydroxy-3-methylglutaryl-CoAreductase (HMG-CoA reductase) polypeptides, mevalonate kinase (MVK)polypeptides, phosphomevalonate kinase (PMK) polypeptides,diphosphomevalonte decarboxylase (MVD) polypeptides, phosphomevalonatedecarboxylase (PMDC) polypeptides, isopentenyl phosphate kinase (IPK)polypeptides, IDI polypeptides, and polypeptides (e.g., fusionpolypeptides) having an activity of two or more MVA pathwaypolypeptides. In particular, MVA pathway polypeptides includepolypeptides, fragments of polypeptides, peptides, and fusionspolypeptides that have at least one activity of an MVA pathwaypolypeptide. Exemplary MVA pathway nucleic acids include nucleic acidsthat encode a polypeptide, fragment of a polypeptide, peptide, or fusionpolypeptide that has at least one activity of an MVA pathwaypolypeptide. Exemplary MVA pathway polypeptides and nucleic acidsinclude naturally-occurring polypeptides and nucleic acids from any ofthe source organisms described herein as well as mutant polypeptides andnucleic acids derived from any of the source organisms described herein.

In particular, acetyl-CoA acetyltransferase polypeptides (AA-CoAthiolase or AACT) convert two molecules of acetyl-CoA intoacetoacetyl-CoA. Standard methods (such as those described herein) canbe used to determine whether a polypeptide has AA-CoA thiolasepolypeptide activity by measuring the ability of the polypeptide toconvert two molecules of acetyl-CoA into acetoacetyl-CoA in vitro, in acell extract, or in vivo. 3-hydroxy-3-methylglutaryl-CoA synthase(HMG-CoA synthase or HMGS) polypeptides convert acetoacetyl-CoA into3-hydroxy-3-methylglutaryl-CoA. Standard methods (such as thosedescribed herein) can be used to determine whether a polypeptide hasHMG-CoA synthase polypeptide activity by measuring the ability of thepolypeptide to convert acetoacetyl-CoA into3-hydroxy-3-methylglutaryl-CoA in vitro, in a cell extract, or in vivo.

3-hydroxy-3-methylglutaryl-CoA reductase (HMG-CoA reductase or HMGR)polypeptides convert 3-hydroxy-3-methylglutaryl-CoA into mevalonate.Standard methods (such as those described herein) can be used todetermine whether a polypeptide has HMG-CoA reductase polypeptideactivity by measuring the ability of the polypeptide to convert3-hydroxy-3-methylglutaryl-CoA into mevalonate in vitro, in a cellextract, or in vivo. Mevalonate kinase (MVK) polypeptide phosphorylatesmevalonate to form mevalonate-5-phosphate. Standard methods (such asthose described herein) can be used to determine whether a polypeptidehas MVK polypeptide activity by measuring the ability of the polypeptideto convert mevalonate into mevalonate-5-phosphate in vitro, in a cellextract, or in vivo.

Phosphomevalonate kinase (PMK) polypeptides phosphorylatesmevalonate-5-phosphate to form mevalonate-5-diphosphate. Standardmethods (such as those described herein) can be used to determinewhether a polypeptide has PMK polypeptide activity by measuring theability of the polypeptide to convert mevalonate-5-phosphate intomevalonate-5-diphosphate in vitro, in a cell extract, or in vivo.

Diphosphomevalonte decarboxylase (MVD or DPMDC) polypeptides convertmevalonate-5-diphosphate into isopentenyl diphosphate (IPP). Standardmethods (such as those described herein) can be used to determinewhether a polypeptide has MVD polypeptide activity by measuring theability of the polypeptide to convert mevalonate-5-diphosphate into IPPin vitro, in a cell extract, or in vivo.

Phosphomevalonate decarboxylase (PMDC) polypeptides convertmevalonate-5-phosphate into isopentenyl phosphate (IP). Standard methods(such as those described herein) can be used to determine whether apolypeptide has PMDC polypeptide activity by measuring the ability ofthe polypeptide to convert mevalonate-5-phosphate into IP in vitro, in acell extract, or in vivo.

Isopentenyl phosphate kinase (IPK) polypeptides phosphorylate isopentylphosphate (IP) to form isopentenyl diphosphate (IPP). Standard methods(such as those described herein) can be used to determine whether apolypeptide has IPK polypeptide activity by measuring the ability of thepolypeptide to convert IP into IPP in vitro, in a cell extract, or invivo.

Exemplary IDI polypeptides and nucleic acids are described above.

Exemplary Methods for Isolating Nucleic Acids

Isoprene synthase, DXS, IDI, and/or MVA pathway nucleic acids can beisolated using standard methods. Methods of obtaining desired nucleicacids from a source organism of interest (such as a bacterial genome)are common and well known in the art of molecular biology (see, forexample, WO 2004/033646 and references cited therein, which are eachhereby incorporated by reference in their entireties, particularly withrespect to the isolation of nucleic acids of interest). For example, ifthe sequence of the nucleic acid is known (such as any of the knownnucleic acids described herein), suitable genomic libraries may becreated by restriction endonuclease digestion and may be screened withprobes complementary to the desired nucleic acid sequence. Once thesequence is isolated, the DNA may be amplified using standard primerdirected amplification methods such as polymerase chain reaction (PCR)(U.S. Pat. No. 4,683,202, which is incorporated by reference in itsentirety, particularly with respect to PCR methods) to obtain amounts ofDNA suitable for transformation using appropriate vectors.

Alternatively, isoprene synthase, DXS, IDI, and/or MVA pathway nucleicacids (such as any isoprene synthase, DXS, IDI, and/or MVA pathwaynucleic acids with a known nucleic acid sequence) can be chemicallysynthesized using standard methods.

Additional isoprene synthase, DXS, IDI, or MVA pathway polypeptides andnucleic acids which may be suitable for use in the compositions andmethods described herein can be identified using standard methods. Forexample, cosmid libraries of the chromosomal DNA of organisms known toproduce isoprene naturally can be constructed in organisms such as E.coli, and then screened for isoprene production. In particular, cosmidlibraries may be created where large segments of genomic DNA (35-45 kb)are packaged into vectors and used to transform appropriate hosts.Cosmid vectors are unique in being able to accommodate large quantitiesof DNA. Generally cosmid vectors have at least one copy of the cos DNAsequence which is needed for packaging and subsequent circularization ofthe heterologous DNA. In addition to the cos sequence, these vectorsalso contain an origin of replication such as Co1EI and drug resistancemarkers such as a nucleic acid resistant to ampicillin or neomycin.Methods of using cosmid vectors for the transformation of suitablebacterial hosts are well described in Sambrook et al., MolecularCloning: A Laboratory Manual, 2^(nd) ed., Cold Spring Harbor, 1989,which is hereby incorporated by reference in its entirety, particularlywith respect to transformation methods. Typically to clone cosmids,heterologous DNA is isolated using the appropriate restrictionendonucleases and ligated adjacent to the cos region of the cosmidvector using the appropriate ligases. Cosmid vectors containing thelinearized heterologous DNA are then reacted with a DNA packagingvehicle such as bacteriophage. During the packaging process, the cossites are cleaved and the heterologous DNA is packaged into the headportion of the bacterial viral particle. These particles are then usedto transfect suitable host cells such as E. coli. Once injected into thecell, the heterologous DNA circularizes under the influence of the cossticky ends. In this manner, large segments of heterologous DNA can beintroduced and expressed in host cells.

Additional methods for obtaining isoprene synthase, DXS, IDI, and/or MVApathway nucleic acids include screening a metagenomic library by assay(such as the headspace assay described herein) or by PCR using primersdirected against nucleotides encoding for a length of conserved aminoacids (for example, at least 3 conserved amino acids). Conserved aminoacids can be identified by aligning amino acid sequences of knownisoprene synthase, DXS, IDI, and/or MVA pathway polypeptides. Conservedamino acids for isoprene synthase polypeptides can be identified basedon aligned sequences of known isoprene synthase polypeptides. Anorganism found to produce isoprene naturally can be subjected tostandard protein purification methods (which are well known in the art)and the resulting purified polypeptide can be sequenced using standardmethods. Other methods are found in the literature (see, for example,Julsing et al., Applied. Microbiol. Biotechnol. 75: 1377-84, 2007;Withers et al., Appl Environ Microbiol. 73(19):6277-83, 2007, which areeach hereby incorporated by reference in their entireties, particularlywith respect to identification of nucleic acids involved in thesynthesis of isoprene).

Additionally, standard sequence alignment and/or structure predictionprograms can be used to identify additional DXS, IDI, or MVA pathwaypolypeptides and nucleic acids based on the similarity of their primaryand/or predicted polypeptide secondary structure with that of known DXS,IDI, or MVA pathway polypeptides and nucleic acids. Standard databasessuch as the swissprot-trembl database (world-wide web at “expasy.org”,Swiss Institute of Bioinformatics Swiss-Prot group CMU-1 rue MichelServet CH-1211 Geneva 4, Switzerland) can also be used to identifyisoprene synthase, DXS, IDI, or MVA pathway polypeptides and nucleicacids. The secondary and/or tertiary structure of an isoprene synthase,DXS, IDI, or MVA pathway polypeptide can be predicted using the defaultsettings of standard structure prediction programs, such asPredictProtein (630 West, 168 Street, BB217, New York, N.Y. 10032, USA).Alternatively, the actual secondary and/or tertiary structure of anisoprene synthase, DXS, IDI, or MVA pathway polypeptide can bedetermined using standard methods. Additional isoprene synthase, DXS,IDI, or MVA pathway nucleic acids can also be identified byhybridization to probes generated from known isoprene synthase, DXS,IDI, or MVA pathway nucleic acids.

Exemplary Promoters and Vectors

Any of the isoprene synthase, DXS, IDI, or MVA pathway nucleic aciddescribed herein can be included in one or more vectors. Accordingly,the invention also features vectors with one more nucleic acids encodingany of the isoprene synthase, DXS, IDI, or MVA pathway polypeptides thatare described herein. As used herein, a “vector” means a construct thatis capable of delivering, and desirably expressing one or more nucleicacids of interest in a host cell. Examples of vectors include, but arenot limited to, plasmids, viral vectors, DNA or RNA expression vectors,cosmids, and phage vectors. In some embodiments, the vector contains anucleic acid under the control of an expression control sequence.

As used herein, an “expression control sequence” means a nucleic acidsequence that directs transcription of a nucleic acid of interest. Anexpression control sequence can be a promoter, such as a constitutive oran inducible promoter, or an enhancer. An “inducible promoter” is apromoter that is active under environmental or developmental regulation.The expression control sequence is operably linked to the nucleic acidsegment to be transcribed.

In some embodiments, the vector contains a selective marker. The term“selective marker” refers to a nucleic acid capable of expression in ahost cell that allows for ease of selection of those host cellscontaining an introduced nucleic acid or vector. Examples of selectablemarkers include, but are not limited to, antibiotic resistance nucleicacids (e.g., kanamycin, ampicillin, carbenicillin, gentamicin,hygromycin, phleomycin, bleomycin, neomycin, or chloramphenicol) and/ornucleic acids that confer a metabolic advantage, such as a nutritionaladvantage on the host cell. Exemplary nutritional selective markersinclude those markers known in the art as amdS, argB, and pyr4. Markersuseful in vector systems for transformation of Trichoderma are known inthe art (see, e.g., Finkelstein, Chapter 6 in Biotechnology ofFilamentous Fungi, Finkelstein et al., Eds. Butterworth-Heinemann,Boston, Mass., Chap. 6., 1992; and Kinghorn et al., Applied MolecularGenetics of Filamentous Fungi, Blackie Academic and Professional,Chapman and Hall, London, 1992, which are each hereby incorporated byreference in their entireties, particularly with respect to selectivemarkers). In some embodiments, the selective marker is the amdS nucleicacid, which encodes the enzyme acetamidase, allowing transformed cellsto grow on acetamide as a nitrogen source. The use of an A. nidulansamdS nucleic acid as a selective marker is described in Kelley et al.,EMBO J. 4:475-479, 1985 and Penttila et al., Gene 61:155-164, 1987(which are each hereby incorporated by reference in their entireties,particularly with respect to selective markers). In some embodiments, anisoprene synthase, DXS, IDI, or MVA pathway nucleic acid integrates intoa chromosome of the cells without a selective marker.

Suitable vectors are those which are compatible with the host cellemployed. Suitable vectors can be derived, for example, from abacterium, a virus (such as bacteriophage T7 or a M-13 derived phage), acosmid, a yeast, or a plant. Protocols for obtaining and using suchvectors are known to those in the art (see, for example, Sambrook etal., Molecular Cloning: A Laboratory Manual, 2^(nd) ed., Cold SpringHarbor, 1989, which is hereby incorporated by reference in its entirety,particularly with respect to the use of vectors).

Promoters are well known in the art. Any promoter that functions in thehost cell can be used for expression of an isoprene synthase, DXS, IDI,or MVA pathway nucleic acid in the host cell. Initiation control regionsor promoters, which are useful to drive expression of isoprene synthase,DXS, IDI, or MVA pathway nucleic acids in various host cells arenumerous and familiar to those skilled in the art (see, for example, WO2004/033646 and references cited therein, which are each herebyincorporated by reference in their entireties, particularly with respectto vectors for the expression of nucleic acids of interest). Virtuallyany promoter capable of driving these nucleic acids is suitable for thepresent invention including, but not limited to, CYC1, HIS3, GAL1,GAL10, ADH1, PGK, PHO5, GAPDH, ADCI, TRP1, URA3, LEU2, ENO, and TPI(useful for expression in Saccharomyces); AOX1 (useful for expression inPichia); and lac, trp,

P_(L),

P_(R), T7, tac, and trc (useful for expression in E. coli).

In some embodiments, a glucose isomerase promoter is used (see, forexample, U.S. Pat. No. 7,132,527 and references cited therein, which areeach hereby incorporated by reference in their entireties, particularlywith respect promoters and plasmid systems for expressing polypeptidesof interest). Reported glucose isomerase promoter mutants can be used tovary the level of expression of the polypeptide encoded by a nucleicacid operably linked to the glucose isomerase promoter (U.S. Pat. No.7,132,527). In various embodiments, the glucose isomerase promoter iscontained in a low, medium, or high copy plasmid (U.S. Pat. No.7,132,527).

In various embodiments, an isoprene synthase, DXS, IDI, and/or MVApathway nucleic acid is contained in a low copy plasmid (e.g., a plasmidthat is maintained at about 1 to about 4 copies per cell), medium copyplasmid (e.g., a plasmid that is maintained at about 10 to about 15copies per cell), or high copy plasmid (e.g., a plasmid that ismaintained at about 50 or more copies per cell). In some embodiments,the heterologous or extra endogenous isoprene synthase, DXS, IDI, or MVApathway nucleic acid is operably linked to a T7 promoter. In someembodiments, the heterologous or extra endogenous isoprene synthase,DXS, IDI, or MVA pathway nucleic acid operably linked to a T7 promoteris contained in a medium or high copy plasmid. In some embodiments, theheterologous or extra endogenous isoprene synthase, DXS, IDI, or MVApathway nucleic acid is operably linked to a Trc promoter. In someembodiments, the heterologous or extra endogenous isoprene synthase,DXS, IDI, or MVA pathway nucleic acid operably linked to a Trc promoteris contained in a medium or high copy plasmid. In some embodiments, theheterologous or extra endogenous isoprene synthase, DXS, IDI, or MVApathway nucleic acid is operably linked to a Lac promoter. In someembodiments, the heterologous or extra endogenous isoprene synthase,DXS, IDI, or MVA pathway nucleic acid operably linked to a Lac promoteris contained in a low copy plasmid. In some embodiments, theheterologous or extra endogenous isoprene synthase, DXS, IDI, or MVApathway nucleic acid is operably linked to an endogenous promoter, suchas an endogenous Escherichia, Panteoa, Bacillus, Yarrowia, Streptomyces,or Trichoderma promoter or an endogenous alkaline serine protease,isoprene synthase, DXS, IDI, or MVA pathway promoter. In someembodiments, the heterologous or extra endogenous isoprene synthase,DXS, IDI, or MVA pathway nucleic acid operably linked to an endogenouspromoter is contained in a high copy plasmid.

In some embodiments, the vector is a replicating plasmid that does notintegrate into a chromosome in the cells. In some embodiments, part orall of the vector integrates into a chromosome in the cells.

In some embodiments, the vector is any vector which when introduced intoa fungal host cell is integrated into the host cell genome and isreplicated. Reference is made to the Fungal Genetics Stock CenterCatalogue of Strains (FGSC, the world-wide web at “fgsc.net” and thereferences cited therein, which are each hereby incorporated byreference in their entireties, particularly with respect to vectors) fora list of vectors. Additional examples of suitable expression and/orintegration vectors are provided in Sambrook et al., Molecular Cloning:A Laboratory Manual, 2^(nd) ed., Cold Spring Harbor, 1989, CurrentProtocols in Molecular Biology (F. M. Ausubel et al. (eds) 1987,Supplement 30, section 7.7.18); van den Hondel et al. in Bennett andLasure (Eds.) More Gene Manipulations in Fungi, Academic Press pp.396-428, 1991; and U.S. Pat. No. 5,874,276, which are each herebyincorporated by reference in their entireties, particularly with respectto vectors. Particularly useful vectors include pFB6, pBR322, PUC18,pUC100, and pENTR/D.

In some embodiments, an isoprene synthase, DXS, IDI, or MVA pathwaynucleic acid is operably linked to a suitable promoter that showstranscriptional activity in a fungal host cell. The promoter may bederived from one or more nucleic acids encoding a polypeptide that iseither endogenous or heterologous to the host cell. In some embodiments,the promoter is useful in a Trichoderma host. Suitable non-limitingexamples of promoters include cbh1, cbh2, egl1, egl2, pepA, hfb1, hfb2,xyn1, and amy. In some embodiments, the promoter is one that is nativeto the host cell. For example, in some embodiments when T. reesei is thehost, the promoter is a native T. reesei promoter. In some embodiments,the promoter is T. reesei cbh1, which is an inducible promoter and hasbeen deposited in GenBank under Accession No. D86235, which isincorporated by reference in its entirety, particularly with respect topromoters. In some embodiments, the promoter is one that is heterologousto the fungal host cell. Other examples of useful promoters includepromoters from the genes of A. awamori and A. niger glucoamylase (glaA)(Nunberg et al., Mol. Cell. Biol. 4:2306-2315, 1984 and Boel et al.,EMBO J. 3:1581-1585, 1984, which are each hereby incorporated byreference in their entireties, particularly with respect to promoters);Aspergillus niger alpha amylases, Aspergillus oryzae TAKA amylase, T.reesei xln1, and the T. reesei cellobiohydrolase 1 (EP 137280, which isincorporated by reference in its entirety, particularly with respect topromoters).

In some embodiments, the expression vector also includes a terminationsequence. Termination control regions may also be derived from variousgenes native to the host cell. In some embodiments, the terminationsequence and the promoter sequence are derived from the same source. Inanother embodiment, the termination sequence is endogenous to the hostcell. A particularly suitable terminator sequence is cbh1 derived from aTrichoderma strain (such as T. reesei). Other useful fungal terminatorsinclude the terminator from an A. niger or A. awamori glucoamylasenucleic acid (Nunberg et al., Mol. Cell Biol. 4:2306-2315, 1984 and Boelet al., EMBO J. 3:1581-1585, 1984; which are each hereby incorporated byreference in their entireties, particularly with respect to fungalterminators). Optionally, a termination site may be included. Foreffective expression of the polypeptides, DNA encoding the polypeptideare linked operably through initiation codons to selected expressioncontrol regions such that expression results in the formation of theappropriate messenger RNA.

In some embodiments, the promoter, coding, region, and terminator alloriginate from the isoprene synthase, DXS, IDI, or MVA pathway nucleicacid to be expressed. In some embodiments, the coding region for anisoprene synthase, DXS, IDI, or MVA pathway nucleic acid is insertedinto a general-purpose expression vector such that it is under thetranscriptional control of the expression construct promoter andterminator sequences. In some embodiments, genes or part thereof areinserted downstream of the strong cbh1 promoter.

An isoprene synthase, DXS, IDI, or MVA pathway nucleic acid can beincorporated into a vector, such as an expression vector, using standardtechniques (Sambrook et al., Molecular Cloning: A Laboratory Manual,Cold Spring Harbor, 1982, which is hereby incorporated by reference inits entirety, particularly with respect to the screening of appropriateDNA sequences and the construction of vectors). Methods used to ligatethe DNA construct comprising a nucleic acid of interest (such as anisoprene synthase, DXS, IDI, or MVA pathway nucleic acid), a promoter, aterminator, and other sequences and to insert them into a suitablevector are well known in the art. For example, restriction enzymes canbe used to cleave the isoprene synthase, DXS, IDI, or MVA pathwaynucleic acid and the vector. Then, the compatible ends of the cleavedisoprene synthase, DXS, IDI, or MVA pathway nucleic acid and the cleavedvector can be ligated. Linking is generally accomplished by ligation atconvenient restriction sites. If such sites do not exist, the syntheticoligonucleotide linkers are used in accordance with conventionalpractice (see, Sambrook et al., Molecular Cloning: A Laboratory Manual,2^(nd) ed., Cold Spring Harbor, 1989, and Bennett and Lasure, More GeneManipulations in Fungi, Academic Press, San Diego, pp 70-76, 1991, whichare each hereby incorporated by reference in their entireties,particularly with respect to oligonucleotide linkers). Additionally,vectors can be constructed using known recombination techniques (e.g.,Invitrogen Life Technologies, Gateway Technology).

In some embodiments, it may be desirable to over-express isoprenesynthase, DXS, IDI, or MVA pathway nucleic acids at levels far higherthan currently found in naturally-occurring cells. This result may beaccomplished by the selective cloning of the nucleic acids encodingthose polypeptides into multicopy plasmids or placing those nucleicacids under a strong inducible or constitutive promoter. Methods forover-expressing desired polypeptides are common and well known in theart of molecular biology and examples may be found in Sambrook et al.,Molecular Cloning: A Laboratory Manual, 2^(nd) ed., Cold Spring Harbor,1989, which is hereby incorporated by reference in its entirety,particularly with respect to cloning techniques.

The following resources include descriptions of additional generalmethodology useful in accordance with the invention: Kreigler, GeneTransfer and Expression; A Laboratory Manual, 1990 and Ausubel et al.,Eds. Current Protocols in Molecular Biology, 1994, which are each herebyincorporated by reference in their entireties, particularly with respectto molecular biology and cloning techniques.

Exemplary Source Organisms

Isoprene synthase, DXS, IDI, or MVA pathway nucleic acids (and theirencoded polypeptides) can be obtained from any organism that naturallycontains isoprene synthase, DXS, IDI, and/or MVA pathway nucleic acids.As noted above, isoprene is formed naturally by a variety of organisms,such as bacteria, yeast, plants, and animals. Organisms contain the MVApathway, DXP pathway, or both the MVA and DXP pathways for producingisoprene (FIG. 19). Thus, DXS nucleic acids can be obtained, e.g., fromany organism that contains the DXP pathway or contains both the MVA andDXP pathways. IDI and isoprene synthase nucleic acids can be obtained,e.g., from any organism that contains the MVA pathway, DXP pathway, orboth the MVA and DXP pathways. MVA pathway nucleic acids can beobtained, e.g., from any organism that contains the MVA pathway orcontains both the MVA and DXP pathways.

In some embodiments, the nucleic acid sequence of the isoprene synthase,DXS, DI, or MVA pathway nucleic is identical to the sequence of anucleic acid that is produced by any of the following organisms innature. In some embodiments, the amino acid sequence of the isoprenesynthase, DXS, IDI, or MVA pathway polypeptide is identical to thesequence of a polypeptide that is produced by any of the followingorganisms in nature. In some embodiments, the isoprene synthase, DXS,IDI, or MVA pathway nucleic acid or polypeptide is a mutant nucleic acidor polypeptide derived from any of the organisms described herein. Asused herein, “derived from” refers to the source of the nucleic acid orpolypeptide into which one or more mutations is introduced. For example,a polypeptide that is “derived from a plant polypeptide” refers topolypeptide of interest that results from introducing one or moremutations into the sequence of a wild-type (i.e., a sequence occurringin nature) plant polypeptide.

In some embodiments, the source organism is a fungus, examples of whichare species of Aspergillus such as A. oryzae and A. niger, species ofSaccharomyces such as S. cerevisiae, species of Schizosaccharomyces suchas S. pombe, and species of Trichoderma such as T. reesei. In someembodiments, the source organism is a filamentous fungal cell. The term“filamentous fungi” refers to all filamentous forms of the subdivisionEumycotina (see, Alexopoulos, C. J. (1962), Introductory Mycology,Wiley, New York). These fungi are characterized by a vegetative myceliumwith a cell wall composed of chitin, cellulose, and other complexpolysaccharides. The filamentous fungi are morphologically,physiologically, and genetically distinct from yeasts. Vegetative growthby filamentous fungi is by hyphal elongation and carbon catabolism isobligatory aerobic. The filamentous fungal parent cell may be a cell ofa species of, but not limited to, Trichoderma, (e.g., Trichodermareesei, the asexual morph of Hypocrea jecorina, previously classified asT. longibrachiatum, Trichoderma viride, Trichoderma koningii,Trichoderma harzianum) (Sheir-Neirs et al., Appl. Microbiol. Biotechnol20: 46-53, 1984; ATCC No. 56765 and ATCC No. 26921); Penicillium sp.,Humicola sp. (e.g., H. insolens, H. lanuginose, or H. grisea);Chrysosporium sp. (e.g., C. lucknowense), Gliocladium sp., Aspergillussp. (e.g., A. oryzae, A. niger, A sojae, A. japonicus, A. nidulans, orA. awamori) (Ward et al., Appl. Microbiol. Biotechnol. 39: 7380743, 1993and Goedegebuur et al., Genet. 41: 89-98, 2002), Fusarium sp., (e.g., F.roseum, F. graminum F. cerealis, F. oxysporuim, or F. venenatum),Neurospora sp., (e.g., N. crassa), Hypocrea sp., Mucor sp., (e.g., M.miehei), Rhizopus sp. and Emericella sp. (see also, Innis et al., Sci.228: 21-26, 1985). The term “Trichoderma” or “Trichoderma sp.” or“Trichoderma spp.” refer to any fungal genus previously or currentlyclassified as Trichoderma.

In some embodiments, the fungus is A. nidulans, A. awamori, A. oryzae,A. aculeatus, A. niger, A. japonicus, T. reesei, T. viride, F.oxysporum, or F. solani. Aspergillus strains are disclosed in Ward etal., Appl. Microbiol. Biotechnol. 39:738-743, 1993 and Goedegebuur etal., Curr Gene 41:89-98, 2002, which are each hereby incorporated byreference in their entireties, particularly with respect to fungi. Inparticular embodiments, the fungus is a strain of Trichoderma, such as astrain of T. reesei. Strains of T. reesei are known and non-limitingexamples include ATCC No. 13631, ATCC No. 26921, ATCC No. 56764, ATCCNo. 56765, ATCC No. 56767, and NRRL 15709, which are each herebyincorporated by reference in their entireties, particularly with respectto strains of T. reesei. In some embodiments, the host strain is aderivative of RL-P37. RL-P37 is disclosed in Sheir-Neiss et al., Appl.Microbiol. Biotechnology 20:46-53, 1984, which is hereby incorporated byreference in its entirety, particularly with respect to strains of T.reesei.

In some embodiments, the source organism is a yeast, such asSaccharomyces sp., Schizosaccharomyces sp., Pichia sp., or Candida sp.In some embodiments, the source organism is a bacterium, such as strainsof Bacillus such as B. lichenformis or B. subtilis, strains of Pantoeasuch as P. citrea, strains of Pseudomonas such as P. alcaligenes,strains of Streptomyces such as S. albus, S. lividans, or S.rubiginosus, or strains of Escherichia such as E. coli.

As used herein, “the genus Bacillus” includes all species within thegenus “Bacillus,” as known to those of skill in the art, including butnot limited to B. subtilis, B. licheniformis, B. lentus, B. brevis, B.stearothermophilus, B. alkalophilus, B. amyloliquefaciens, B. clausii,B. halodurans, B. megaterium, B. coagulans, B. circulans, B. lautus, andB. thuringiensis. It is recognized that the genus Bacillus continues toundergo taxonomical reorganization. Thus, it is intended that the genusinclude species that have been reclassified, including but not limitedto such organisms as B. stearothermophilus, which is now named“Geobacillus stearothermophilus.” The production of resistant endosporesin the presence of oxygen is considered the defining feature of thegenus Bacillus, although this characteristic also applies to therecently named Alicyclobacillus, Amphibacillus, Aneurinibacillus,Anoxybacillus, Brevibacillus, Filobacillus, Gracilibacillus,Halobacillus, Paenibacillus, Salibacillus, Thermobacillus, Ureibacillus,and Virgibacillus.

In some embodiments, the source organism is a gram-positive bacterium.Non-limiting examples include strains of Streptomyces (e.g., S. albus,S. lividans, S. coelicolor, or S. griseus) and Bacillus. In someembodiments, the source organism is a gram-negative bacterium, such asE. coli or Pseudomonas sp.

In some embodiments, the source organism is a plant, such as a plantfrom the family Fabaceae, such as the Faboideae subfamily. In someembodiments, the source organism is kudzu, poplar (such as Populusalba×tremula CAC35696 or Populus alba) (Sasaki et al., FEBS Letters579(11): 2514-2518, 2005), aspen (such as Populus tremuloides), orQuercus robur.

In some embodiments, the source organism is an algae, such as a greenalgae, red algae, glaucophytes, chlorarachniophytes, euglenids,chromista, or dinoflagellates.

In some embodiments, the source organism is a cyanobacteria, such ascyanobacteria classified into any of the following groups based onmorphology: Chroococcales, Pleurocapsales, Oscillatoriales, Nostocales,or Stigonematales.

Exemplary Host Cells

A variety of host cells can be used to express isoprene synthase, DXS,IDI, and/or MVA pathway polypeptides and to produce isoprene in themethods of the claimed invention. Exemplary host cells include cellsfrom any of the organisms listed in the prior section under the heading“Exemplary Source Organisms.” The host cell may be a cell that naturallyproduces isoprene or a cell that does not naturally produce isoprene. Insome embodiments, the host cell naturally produces isoprene using theDXP pathway, and an isoprene synthase, DXS, and/or IDI nucleic acid isadded to enhance production of isoprene using this pathway. In someembodiments, the host cell naturally produces isoprene using the MVApathway, and an isoprene synthase and/or one or more MVA pathway nucleicacids are added to enhance production of isoprene using this pathway. Insome embodiments, the host cell naturally produces isoprene using theDXP pathway and one or more MVA pathway nucleic acids are added toproduce isoprene using part or all of the MVA pathway as well as the DXPpathway. In some embodiments, the host cell naturally produces isopreneusing both the DXP and MVA pathways and one or more isoprene synthase,DXS, IDI, or MVA pathway nucleic acids are added to enhance productionof isoprene by one or both of these pathways.

Exemplary Transformation Methods

Isoprene synthase, DXS, IDI, and/or MVA pathway nucleic acids or vectorscontaining them can be inserted into a host cell (e.g., a plant cell, afungal cell, a yeast cell, or a bacterial cell described herein) usingstandard techniques for expression of the encoded isoprene synthase,DXS, IDI, and/or MVA pathway polypeptide. Introduction of a DNAconstruct or vector into a host cell can be performed using techniquessuch as transformation, electroporation, nuclear microinjection,transduction, transfection (e.g., lipofection mediated or DEAE-Dextrinmediated transfection or transfection using a recombinant phage virus),incubation with calcium phosphate DNA precipitate, high velocitybombardment with DNA-coated microprojectiles, and protoplast fusion.General transformation techniques are known in the art (see, e.g.,Current Protocols in Molecular Biology (F. M. Ausubel et al. (eds)Chapter 9, 1987; Sambrook et al., Molecular Cloning: A LaboratoryManual, 2^(nd) ed., Cold Spring Harbor, 1989; and Campbell et al., Curr.Genet. 16:53-56, 1989, which are each hereby incorporated by referencein their entireties, particularly with respect to transformationmethods). The expression of heterologous polypeptide in Trichoderma isdescribed in U.S. Pat. Nos. 6,022,725; 6,268,328; 7,262,041; WO2005/001036; Harkki et al.; Enzyme Microb. Technol. 13:227-233, 1991;Harkki et al., Bio Technol. 7:596-603, 1989; EP 244,234; EP 215,594; andNevalainen et al., “The Molecular Biology of Trichoderma and itsApplication to the Expression of Both Homologous and HeterologousGenes,” in Molecular Industrial Mycology, Eds. Leong and Berka, MarcelDekker Inc., NY pp. 129-148, 1992, which are each hereby incorporated byreference in their entireties, particularly with respect totransformation and expression methods). Reference is also made to Cao etal., (Sci. 9:991-1001, 2000; EP 238023; and Yelton et al., Proceedings.Natl. Acad. Sci. USA 81:1470-1474, 1984 (which are each herebyincorporated by reference in their entireties, particularly with respectto transformation methods) for transformation of Aspergillus strains.The introduced nucleic acids may be integrated into chromosomal DNA ormaintained as extrachromosomal replicating sequences.

Any method known in the art may be used to select transformants. In onenon-limiting example, stable transformants including an amdS marker aredistinguished from unstable transformants by their faster growth rateand the formation of circular colonies with a smooth, rather than raggedoutline on solid culture medium containing acetamide. Additionally, insome cases a further test of stability is conducted by growing thetransformants on a solid non-selective medium (e.g., a medium that lacksacetamide), harvesting spores from this culture medium, and determiningthe percentage of these spores which subsequently germinate and grow onselective medium containing acetamide.

In some embodiments, fungal cells are transformed by a process involvingprotoplast formation and transformation of the protoplasts followed byregeneration of the cell wall in a known manner. In one specificembodiment, the preparation of Trichoderma sp. for transformationinvolves the preparation of protoplasts from fungal mycelia (see,Campbell et al., Curr. Genet. 16:53-56, 1989, which is incorporated byreference in its entirety, particularly with respect to transformationmethods). In some embodiments, the mycelia are obtained from germinatedvegetative spores. The mycelia are treated with an enzyme that digeststhe cell wall resulting in protoplasts. The protoplasts are thenprotected by the presence of an osmotic stabilizer in the suspendingmedium. These stabilizers include sorbitol, mannitol, potassiumchloride, magnesium sulfate, and the like. Usually the concentration ofthese stabilizers varies between 0.8 M and 1.2 M. It is desirable to useabout a 1.2 M solution of sorbitol in the suspension medium.

Uptake of DNA into the host Trichoderma sp. strain is dependent upon thecalcium ion concentration. Generally, between about 10 mM CaCl₂ and 50mM CaCl₂ is used in an uptake solution. In addition to the calcium ionin the uptake solution, other compounds generally included are abuffering system such as TE buffer (10 Mm Tris, pH 7.4; 1 mM EDTA) or 10mM MOPS, pH 6.0 buffer (morpholinepropanesulfonic acid) and polyethyleneglycol (PEG). While not intending to be bound to any particular theory,it is believed that the polyethylene glycol acts to fuse the cellmembranes, thus permitting the contents of the medium to be deliveredinto the cytoplasm of the Trichoderma sp. strain and the plasmid DNA tobe transferred to the nucleus. This fusion frequently leaves multiplecopies of the plasmid DNA integrated into the host chromosome.

Usually a suspension containing the Trichoderma sp. protoplasts or cellsthat have been subjected to a permeability treatment at a density of 10⁵to 10⁷/mL (such as 2×10⁶/mL) are used in the transformation. A volume of100 μL of these protoplasts or cells in an appropriate solution (e.g.,1.2 M sorbitol and 50 mM CaCl₂) are mixed with the desired DNA.Generally, a high concentration of PEG is added to the uptake solution.From 0.1 to 1 volume of 25% PEG 4000 can be added to the protoplastsuspension. In some embodiments, about 0.25 volumes are added to theprotoplast suspension. Additives such as dimethyl sulfoxide, heparin,spermidine, potassium chloride, and the like may also be added to theuptake solution and aid in transformation. Similar procedures areavailable for other fungal host cells (see, e.g., U.S. Pat. Nos.6,022,725 and 6,268,328, which are each hereby incorporated by referencein their entireties, particularly with respect to transformationmethods).

Generally, the mixture is then cultured at approximately 0° C. for aperiod of between 10 to 30 minutes. Additional PEG is then added to themixture to further enhance the uptake of the desired nucleic acidsequence. The 25% PEG 4000 is generally added in volumes of 5 to 15times the volume of the transformation mixture; however, greater andlesser volumes may be suitable. The 25% PEG 4000 is desirably about 10times the volume of the transformation mixture. After the PEG is added,the transformation mixture is then cultured either at room temperatureor on ice before the addition of a sorbitol and CaCl₂ solution. Theprotoplast suspension is then further added to molten aliquots of agrowth medium. When the growth medium includes a growth selection (e.g.,acetamide or an antibiotic) it permits the growth of transformants only.

The transformation of bacterial cells may be performed according toconventional methods, e.g., as described in Sambrook et al., MolecularCloning: A Laboratory Manual, Cold Spring Harbor, 1982, which is herebyincorporated by reference in its entirety, particularly with respect totransformation methods.

Exemplary Cell Culture Media

The invention also includes a cell or a population of cells in culturethat produce isoprene. By “cells in culture” is meant two or more cellsin a solution (e.g., a cell medium) that allows the cells to undergo oneor more cell divisions. “Cells in culture” do not include plant cellsthat are part of a living, multicellular plant containing cells thathave differentiated into plant tissues. In various embodiments, the cellculture includes at least or about 10, 20, 50, 100, 200, 500, 1,000,5,000, 10,000 or more cells.

Any carbon source can be used to cultivate the host cells. The term“carbon source” refers to one or more carbon-containing compoundscapable of being metabolized by a host cell or organism. For example,the cell medium used to cultivate the host cells may include any carbonsource suitable for maintaining the viability or growing the host cells.

In some embodiments, the carbon source is a carbohydrate (such asmonosaccharide, disaccharide, oligosaccharide, or polysaccharids),invert sugar (e.g., enzymatically treated sucrose syrup), glycerol,glycerine (e.g., a glycerine byproduct of a biodiesel or soap-makingprocess), dihydroxyacetone, one-carbon source, oil (e.g., a plant orvegetable oil such as corn, palm, or soybean oil), acetate, animal fat,animal oil, fatty acid (e.g., a saturated fatty acid, unsaturated fattyacid, or polyunsaturated fatty acid), lipid, phospholipid, glycerolipid,monoglyceride, diglyceride, triglyceride, polypeptide (e.g., a microbialor plant protein or peptide), renewable carbon source (e.g., a biomasscarbon source such as a hydrolyzed biomass carbon source), yeastextract, component from a yeast extract, polymer, acid, alcohol,aldehyde, ketone, amino acid, succinate, lactate, acetate, ethanol, orany combination of two or more of the foregoing. In some embodiments,the carbon source is a product of photosynthesis, including, but notlimited to, glucose. In some embodiment, the carbohydrate is xylose orglucose.

Exemplary monosaccharides include glucose and fructose; exemplaryoligosaccharides include lactose and sucrose, and exemplarypolysaccharides include starch and cellulose. Exemplary carbohydratesinclude C6 sugars (e.g., fructose, mannose, galactose, or glucose) andC5 sugars (e.g., xylose or arabinose). In some embodiments, the cellmedium includes a carbohydrate as well as a carbon source other than acarbohydrate (e.g., glycerol, glycerine, dihydroxyacetone, one-carbonsource, oil, animal fat, animal oil, fatty acid, lipid, phospholipid,glycerolipid, monoglyceride, diglyceride, triglyceride, renewable carbonsource, or a component from a yeast extract). In some embodiments, thecell medium includes a carbohydrate as well as a polypeptide (e.g., amicrobial or plant protein or peptide). In some embodiments, themicrobial polypeptide is a polypeptide from yeast or bacteria. In someembodiments, the plant polypeptide is a polypeptide from soy, corn,canola, jatropha, palm, peanut, sunflower, coconut, mustard, rapeseed,cottonseed, palm kernel, olive, safflower, sesame, or linseed.

In some embodiments, the concentration of the carbohydrate is at leastor about 5 grams per liter of broth (g/L, wherein the volume of brothincludes both the volume of the cell medium and the volume of thecells), such as at least or about 10, 15, 20, 30, 40, 50, 60, 80, 100,150, 200, 300, 400, or more g/L. In some embodiments, the concentrationof the carbohydrate is between about 50 and about 400 g/L, such asbetween about 100 and about 360 g/L, between about 120 and about 360g/L, or between about 200 and about 300 g/L. In some embodiments, thisconcentration of carbohydrate includes the total amount of carbohydratethat is added before and/or during the culturing of the host cells.

Exemplary lipids are any substance containing one or more fatty acidsthat are C4 and above fatty acids that are saturated, unsaturated, orbranched.

Exemplary oils are lipids that are liquid at room temperature. In someembodiments, the lipid contains one or more C4 or above fatty acids(e.g., contains one or more saturated, unsaturated, or branched fattyacid with four or more carbons). In some embodiments, the oil isobtained from soy, corn, canola, jatropha, palm, peanut, sunflower,coconut, mustard, rapeseed, cottonseed, palm kernel, olive, safflower,sesame, linseed, oleagineous microbial cells, Chinese tallow, or anycombination of two or more of the foregoing.

Exemplary fatty acids include compounds of the formula RCOOH, where “R”is a hydrocarbon. Exemplary unsaturated fatty acids include compoundswhere “R” includes at least one carbon-carbon double bond. Exemplaryunsaturated fatty acids include, but are not limited to, oleic acid,vaccenic acid, linoleic acid, palmitelaidic acid, and arachidonic acid.Exemplary polyunsaturated fatty acids include compounds where “R”includes a plurality of carbon-carbon double bonds. Exemplary saturatedfatty acids include compounds where “R” is a saturated aliphatic group.In some embodiments, the carbon source includes one or more C₁₂-C₂₂fatty acids, such as a C₁₂ saturated fatty acid, a C₁₄ saturated fattyacid, a C₁₆ saturated fatty acid, a C₁₈ saturated fatty acid, a C₂₀saturated fatty acid, or a C₂₂ saturated fatty acid. In an exemplaryembodiment, the fatty acid is palmitic acid. In some embodiments, thecarbon source is a salt of a fatty acid (e.g., an unsaturated fattyacid), a derivative of a fatty acid (e.g., an unsaturated fatty acid),or a salt of a derivative of fatty acid (e.g., an unsaturated fattyacid). Suitable salts include, but are not limited to, lithium salts,potassium salts, sodium salts, and the like. Di- and triglycerols arefatty acid esters of glycerol.

In some embodiments, the concentration of the lipid, oil, fat, fattyacid, monoglyceride, diglyceride, or triglyceride is at least or about 1gram per liter of broth (g/L, wherein the volume of broth includes boththe volume of the cell medium and the volume of the cells), such as atleast or about 5, 10, 15, 20, 30, 40, 50, 60, 80, 100, 150, 200, 300,400, or more g/L. In some embodiments, the concentration of the lipid,oil, fat, fatty acid, monoglyceride, diglyceride, or triglyceride isbetween about 10 and about 400 g/L, such as between about 25 and about300 g/L, between about 60 and about 180 g/L, or between about 75 andabout 150 g/L. In some embodiments, the concentration includes the totalamount of the lipid, oil, fat, fatty acid, monoglyceride, diglyceride,or triglyceride that is added before and/or during the culturing of thehost cells. In some embodiments, the carbon source includes both (i) alipid, oil, fat, fatty acid, monoglyceride, diglyceride, or triglycerideand (ii) a carbohydrate, such as glucose. In some embodiments, the ratioof the lipid, oil, fat, fatty acid, monoglyceride, diglyceride, ortriglyceride to the carbohydrate is about 1:1 on a carbon basis (i.e.,one carbon in the lipid, oil, fat, fatty acid, monoglyceride,diglyceride, or triglyceride per carbohydrate carbon). In particularembodiments, the amount of the lipid, oil, fat, fatty acid,monoglyceride, diglyceride, or triglyceride is between about 60 and 180g/L, and the amount of the carbohydrate is between about 120 and 360g/L.

Exemplary microbial polypeptide carbon sources include one or morepolypeptides from yeast or bacteria. Exemplary plant polypeptide carbonsources include one or more polypeptides from soy, corn, canola,jatropha, palm, peanut, sunflower, coconut, mustard, rapeseed,cottonseed, palm kernel, olive, safflower, sesame, or linseed.

Exemplary renewable carbon sources include cheese whey permeate,cornsteep liquor, sugar beet molasses, barley malt, and components fromany of the foregoing. Exemplary renewable carbon sources also includeacetate, glucose, hexose, pentose and xylose present in biomass, such ascorn, switchgrass, sugar cane, cell waste of fermentation processes, andprotein by-product from the milling of soy, corn, or wheat. In someembodiments, the biomass carbon source is a lignocellulosic,hemicellulosic, or cellulosic material such as, but are not limited to,a grass, wheat, wheat straw, bagasse, sugar cane bagasse, soft woodpulp, corn, corn cob or husk, corn kernel, fiber from corn kernels, cornstover, switch grass, rice hull product, or a by-product from wet or drymilling of grains (e.g., corn, sorghum, rye, triticate, barley, wheat,and/or distillers grains). Exemplary cellulosic materials include wood,paper and pulp waste, herbaceous plants, and fruit pulp. In someembodiments, the carbon source includes any plant part, such as stems,grains, roots, or tubers. In some embodiments, all or part of any of thefollowing plants are used as a carbon source: corn, wheat, rye, sorghum,triticate, rice, millet, barley, cassaya, legumes, such as beans andpeas, potatoes, sweet potatoes, bananas, sugarcane, and/or tapioca. Insome embodiments, the carbon source is a biomass hydrolysate, such as abiomass hydrolysate that includes both xylose and glucose or thatincludes both sucrose and glucose.

In some embodiments, the renewable carbon source (such as biomass) ispretreated before it is added to the cell culture medium. In someembodiments, the pretreatment includes enzymatic pretreatment, chemicalpretreatment, or a combination of both enzymatic and chemicalpretreatment (see, for example, Farzaneh et al., Bioresource Technology96 (18): 2014-2018, 2005; U.S. Pat. Nos. 6,176,176; 6,106,888; which areeach hereby incorporated by reference in their entireties, particularlywith respect to the pretreatment of renewable carbon sources). In someembodiments, the renewable carbon source is partially or completelyhydrolyzed before it is added to the cell culture medium.

In some embodiments, the renewable carbon source (such as corn stover)undergoes ammonia fiber expansion (AFEX) pretreatment before it is addedto the cell culture medium (see, for example, Farzaneh et al.,Bioresource Technology 96 (18): 2014-2018, 2005). During AFEXpretreatment, a renewable carbon source is treated with liquid anhydrousammonia at moderate temperatures (such as about 60 to about 100° C.) andhigh pressure (such as about 250 to about 300 psi) for about 5 minutes.Then, the pressure is rapidly released. In this process, the combinedchemical and physical effects of lignin solubilization, hemicellulosehydrolysis, cellulose decrystallization, and increased surface areaenables near complete enzymatic conversion of cellulose andhemicellulose to fermentable sugars. AFEX pretreatment has the advantagethat nearly all of the ammonia can be recovered and reused, while theremaining serves as nitrogen source for microbes in downstreamprocesses. Also, a wash stream is not required for AFEX pretreatment.Thus, dry matter recovery following the AFEX treatment is essentially100%. AFEX is basically a dry to dry process. The treated renewablecarbon source is stable for long periods and can be fed at very highsolid loadings in enzymatic hydrolysis or fermentation processes.Cellulose and hemicellulose are well preserved in the AFEX process, withlittle or no degradation. There is no need for neutralization prior tothe enzymatic hydrolysis of a renewable carbon source that has undergoneAFEX pretreatment. Enzymatic hydrolysis of AFEX-treated carbon sourcesproduces clean sugar streams for subsequent fermentation use.

In some embodiments, the concentration of the carbon source (e.g., arenewable carbon source) is equivalent to at least or about 0.1, 0.5, 1,1.5 2, 3, 4, 5, 10, 15, 20, 30, 40, or 50% glucose (w/v). The equivalentamount of glucose can be determined by using standard HPLC methods withglucose as a reference to measure the amount of glucose generated fromthe carbon source. In some embodiments, the concentration of the carbonsource (e.g., a renewable carbon source) is equivalent to between about0.1 and about 20% glucose, such as between about 0.1 and about 10%glucose, between about 0.5 and about 10% glucose, between about 1 andabout 10% glucose, between about 1 and about 5% glucose, or betweenabout 1 and about 2% glucose.

In some embodiments, the carbon source includes yeast extract or one ormore components of yeast extract. In some embodiments, the concentrationof yeast extract is at least 1 gram of yeast extract per liter of broth(g/L, wherein the volume of broth includes both the volume of the cellmedium and the volume of the cells), such at least or about 5, 10, 15,20, 30, 40, 50, 60, 80, 100, 150, 200, 300, or more g/L. In someembodiments, the concentration of yeast extract is between about 1 andabout 300 g/L, such as between about 1 and about 200 g/L, between about5 and about 200 g/L, between about 5 and about 100 g/L, or between about5 and about 60 g/L. In some embodiments, the concentration includes thetotal amount of yeast extract that is added before and/or during theculturing of the host cells. In some embodiments, the carbon sourceincludes both yeast extract (or one or more components thereof) andanother carbon source, such as glucose. In some embodiments, the ratioof yeast extract to the other carbon source is about 1:5, about 1:10, orabout 1:20 (w/w).

Additionally the carbon source may also be one-carbon substrates such ascarbon dioxide, or methanol. Glycerol production from single carbonsources (e.g., methanol, formaldehyde, or formate) has been reported inmethylotrophic yeasts (Yamada et al., Agric. Biol. Chem., 53(2) 541-543,1989, which is hereby incorporated by reference in its entirety,particularly with respect to carbon sources) and in bacteria (Hunter et.al., Biochemistry, 24, 4148-4155, 1985, which is hereby incorporated byreference in its entirety, particularly with respect to carbon sources).These organisms can assimilate single carbon compounds, ranging inoxidation state from methane to formate, and produce glycerol. Thepathway of carbon assimilation can be through ribulose monophosphate,through serine, or through xylulose-momophosphate (Gottschalk, BacterialMetabolism, Second Edition, Springer-Verlag: New York, 1986, which ishereby incorporated by reference in its entirety, particularly withrespect to carbon sources). The ribulose monophosphate pathway involvesthe condensation of formate with ribulose-5-phosphate to form a sixcarbon sugar that becomes fructose and eventually the three carbonproduct glyceraldehyde-3-phosphate. Likewise, the serine pathwayassimilates the one-carbon compound into the glycolytic pathway viamethylenetetrahydrofolate.

In addition to one and two carbon substrates, methylotrophic organismsare also known to utilize a number of other carbon containing compoundssuch as methylamine, glucosamine and a variety of amino acids formetabolic activity. For example, methylotrophic yeast are known toutilize the carbon from methylamine to form trehalose or glycerol(Bellion et al., Microb. Growth Cl Compd., [Int. Symp.], 7^(th) ed.,415-32. Editors: Murrell et al., Publisher: Intercept, Andover, UK,1993, which is hereby incorporated by reference in its entirety,particularly with respect to carbon sources). Similarly, various speciesof Candida metabolize alanine or oleic acid (Sulter et al., Arch.Microbiol. 153(5), 485-9, 1990, which is hereby incorporated byreference in its entirety, particularly with respect to carbon sources).

In some embodiments, cells are cultured in a standard medium containingphysiological salts and nutrients (see, e.g., Pourquie, J. et al.,Biochemistry and Genetics of Cellulose Degradation, eds. Aubert et al.,Academic Press, pp. 71-86, 1988 and Ilmen et al., Appl. Environ.Microbiol. 63:1298-1306, 1997, which are each hereby incorporated byreference in their entireties, particularly with respect to cell media).Exemplary growth media are common commercially prepared media such asLuria Bertani (LB) broth, Sabouraud Dextrose (SD) broth, or Yeast medium(YM) broth. Other defined or synthetic growth media may also be used,and the appropriate medium for growth of particular host cells are knownby someone skilled in the art of microbiology or fermentation science.

In addition to an appropriate carbon source, the cell medium desirablycontains suitable minerals, salts, cofactors, buffers, and othercomponents known to those skilled in the art suitable for the growth ofthe cultures or the enhancement of isoprene production (see, forexample, WO 2004/033646 and references cited therein and WO 96/35796 andreferences cited therein, which are each hereby incorporated byreference in their entireties, particularly with respect cell medias andcell culture conditions). In some embodiments where an isoprenesynthase, DXS, IDI, and/or MVA pathway nucleic acid is under the controlof an inducible promoter, the inducing agent (e.g., a sugar, metal saltor antimicrobial), is desirably added to the medium at a concentrationeffective to induce expression of an isoprene synthase, DXS, IDI, and/orMVA pathway polypeptide. In some embodiments, cell medium has anantibiotic (such as kanamycin) that corresponds to the antibioticresistance nucleic acid (such as a kanamycin resistance nucleic acid) ona vector that has one or more DXS, IDI, or MVA pathway nucleic acids.

Exemplary Cell Culture Conditions

Materials and methods suitable for the maintenance and growth ofbacterial cultures are well known in the art. Exemplary techniques maybe found in Manual of Methods for General Bacteriology Gerhardt et al.,eds), American Society for Microbiology, Washington, D.C. (1994) orBrock in Biotechnology: A Textbook of Industrial Microbiology, SecondEdition (1989) Sinauer Associates, Inc., Sunderland, Mass., which areeach hereby incorporated by reference in their entireties, particularlywith respect to cell culture techniques. In some embodiments, the cellsare cultured in a culture medium under conditions permitting theexpression of one or more isoprene synthase, DXS, IDI, or MVA pathwaypolypeptides encoded by a nucleic acid inserted into the host cells.

Standard cell culture conditions can be used to culture the cells (see,for example, WO 2004/033646 and references cited therein, which are eachhereby incorporated by reference in their entireties, particularly withrespect to cell culture and fermentation conditions). Cells are grownand maintained at an appropriate temperature, gas mixture, and pH (suchas at about 20 to about 37° C., at about 6% to about 84% CO₂, and at apH between about 5 to about 9). In some embodiments, cells are grown at35° C. in an appropriate cell medium. In some embodiments, e.g.,cultures are cultured at approximately 28° C. in appropriate medium inshake cultures or fermentors until desired amount of isoprene productionis achieved. In some embodiments, the pH ranges for fermentation arebetween about pH 5.0 to about pH 9.0 (such as about pH 6.0 to about pH8.0 or about 6.5 to about 7.0). Reactions may be performed underaerobic, anoxic, or anaerobic conditions based on the requirements ofthe host cells. Exemplary culture conditions for a given filamentousfungus are known in the art and may be found in the scientificliterature and/or from the source of the fungi such as the American TypeCulture Collection and Fungal Genetics Stock Center.

In various embodiments, the cells are grown using any known mode offermentation, such as batch, fed-batch, or continuous processes. In someembodiments, a batch method of fermentation is used. Classical batchfermentation is a closed system where the composition of the media isset at the beginning of the fermentation and is not subject toartificial alterations during the fermentation. Thus, at the beginningof the fermentation the cell medium is inoculated with the desired hostcells and fermentation is permitted to occur adding nothing to thesystem. Typically, however, “batch” fermentation is batch with respectto the addition of carbon source and attempts are often made atcontrolling factors such as pH and oxygen concentration. In batchsystems, the metabolite and biomass compositions of the system changeconstantly until the time the fermentation is stopped. Within batchcultures, cells moderate through a static lag phase to a high growth logphase and finally to a stationary phase where growth rate is diminishedor halted. In some embodiments, cells in log phase are responsible forthe bulk of the isoprene production. In some embodiments, cells instationary phase produce isoprene.

In some embodiments, a variation on the standard batch system is used,such as the Fed-Batch system. Fed-Batch fermentation processes comprisea typical batch system with the exception that the carbon source isadded in increments as the fermentation progresses. Fed-Batch systemsare useful when catabolite repression is apt to inhibit the metabolismof the cells and where it is desirable to have limited amounts of carbonsource in the cell medium. Fed-batch fermentations may be performed withthe carbon source (e.g., glucose) in a limited or excess amount.Measurement of the actual carbon source concentration in Fed-Batchsystems is difficult and is therefore estimated on the basis of thechanges of measurable factors such as pH, dissolved oxygen, and thepartial pressure of waste gases such as CO₂. Batch and Fed-Batchfermentations are common and well known in the art and examples may befound in Brock, Biotechnology: A Textbook of Industrial Microbiology,Second Edition (1989) Sinauer Associates, Inc., which is herebyincorporated by reference in its entirety, particularly with respect tocell culture and fermentation conditions.

In some embodiments, continuous fermentation methods are used.Continuous fermentation is an open system where a defined fermentationmedium is added continuously to a bioreactor and an equal amount ofconditioned medium is removed simultaneously for processing. Continuousfermentation generally maintains the cultures at a constant high densitywhere cells are primarily in log phase growth.

Continuous fermentation allows for the modulation of one factor or anynumber of factors that affect cell growth or isoprene production. Forexample, one method maintains a limiting nutrient such as the carbonsource or nitrogen level at a fixed rate and allows all other parametersto moderate. In other systems, a number of factors affecting growth canbe altered continuously while the cell concentration (e.g., theconcentration measured by media turbidity) is kept constant. Continuoussystems strive to maintain steady state growth conditions. Thus, thecell loss due to media being drawn off is balanced against the cellgrowth rate in the fermentation. Methods of modulating nutrients andgrowth factors for continuous fermentation processes as well astechniques for maximizing the rate of product formation are well knownin the art of industrial microbiology and a variety of methods aredetailed by Brock, Biotechnology: A Textbook of Industrial Microbiology,Second Edition (1989) Sinauer Associates, Inc., which is herebyincorporated by reference in its entirety, particularly with respect tocell culture and fermentation conditions.

In some embodiments, cells are immobilized on a substrate as whole cellcatalysts and subjected to fermentation conditions for isopreneproduction.

In some embodiments, bottles of liquid culture are placed in shakers inorder to introduce oxygen to the liquid and maintain the uniformity ofthe culture. In some embodiments, an incubator is used to control thetemperature, humidity, shake speed, and/or other conditions in which aculture is grown. The simplest incubators are insulated boxes with anadjustable heater, typically going up to ˜65° C. More elaborateincubators can also include the ability to lower the temperature (viarefrigeration), or the ability to control humidity or CO₂ levels. Mostincubators include a timer; some can also be programmed to cycle throughdifferent temperatures, humidity levels, etc. Incubators can vary insize from tabletop to units the size of small rooms.

If desired, a portion or all of the cell medium can be changed toreplenish nutrients and/or avoid the build up of potentially harmfulmetabolic byproducts and dead cells. In the case of suspension cultures,cells can be separated from the media by centrifuging or filtering thesuspension culture and then resuspending the cells in fresh media. Inthe case of adherent cultures, the media can be removed directly byaspiration and replaced. In some embodiments, the cell medium allows atleast a portion of the cells to divide for at least or about 5, 10, 20,40, 50, 60, 65, or more cell divisions in a continuous culture (such asa continuous culture without dilution).

In some embodiments, a constitutive or leaky promoter (such as a Trcpromoter) is used and a compound (such as IPTG) is not added to induceexpression of the isoprene synthase, DXS, IDI, or MVA pathway nucleicacid(s) operably linked to the promoter. In some embodiments, a compound(such as IPTG) is added to induce expression of the isoprene synthase,DXS, IDI, or MVA pathway nucleic acid(s) operably linked to thepromoter.

Exemplary Production of Isoprene

In some embodiments, the cells are cultured in a culture medium underconditions permitting the production of isoprene by the cells. By “peakabsolute productivity” is meant the maximum absolute amount of isoprenein the off-gas during the culturing of cells for a particular period oftime (e.g., the culturing of cells during a particular fermentationrun). By “peak absolute productivity time point” is meant the time pointduring a fermentation run when the absolute amount of isoprene in theoff-gas is at a maximum during the culturing of cells for a particularperiod of time (e.g., the culturing of cells during a particularfermentation run). In some embodiments, the isoprene amount is measuredat the peak absolute productivity time point. In some embodiments, thepeak absolute productivity for the cells is about any of the isopreneamounts disclosed herein.

By “peak specific productivity” is meant the maximum amount of isopreneproduced per cell during the culturing of cells for a particular periodof time (e.g., the culturing of cells during a particular fermentationrun). By “peak specific productivity time point” is meant the time pointduring the culturing of cells for a particular period of time (e.g., theculturing of cells during a particular fermentation run) when the amountof isoprene produced per cell is at a maximum. The peak specificproductivity is determined by dividing the total productivity by theamount of cells, as determined by optical density at 600 nm (OD₆₀₀). Insome embodiments, the isoprene amount is measured at the peak specificproductivity time point. In some embodiments, the peak specificproductivity for the cells is about any of the isoprene amounts per celldisclosed herein.

By “peak volumetric productivity” is meant the maximum amount ofisoprene produced per volume of broth (including the volume of the cellsand the cell medium) during the culturing of cells for a particularperiod of time (e.g., the culturing of cells during a particularfermentation run). By “peak specific volumetric productivity time point”is meant the time point during the culturing of cells for a particularperiod of time (e.g., the culturing of cells during a particularfermentation run) when the amount of isoprene produced per volume ofbroth is at a maximum. The peak specific volumetric productivity isdetermined by dividing the total productivity by the volume of broth andamount of time. In some embodiments, the isoprene amount is measured atthe peak specific volumetric productivity time point. In someembodiments, the peak specific volumetric productivity for the cells isabout any of the isoprene amounts per volume per time disclosed herein.

By “peak concentration” is meant the maximum amount of isoprene producedduring the culturing of cells for a particular period of time (e.g., theculturing of cells during a particular fermentation run). By “peakconcentration time point” is meant the time point during the culturingof cells for a particular period of time (e.g., the culturing of cellsduring a particular fermentation run) when the amount of isopreneproduced per cell is at a maximum. In some embodiments, the isopreneamount is measured at the peak concentration time point. In someembodiments, the peak concentration for the cells is about any of theisoprene amounts disclosed herein.

By “average volumetric productivity” is meant the average amount ofisoprene produced per volume of broth (including the volume of the cellsand the cell medium) during the culturing of cells for a particularperiod of time (e.g., the culturing of cells during a particularfermentation run). The average volumetric productivity is determined bydividing the total productivity by the volume of broth and amount oftime. In some embodiments, the average specific volumetric productivityfor the cells is about any of the isoprene amounts per volume per timedisclosed herein.

By “cumulative total productivity” is meant the cumulative, total amountof isoprene produced during the culturing of cells for a particularperiod of time (e.g., the culturing of cells during a particularfermentation run). In some embodiments, the cumulative, total amount ofisoprene is measured. In some embodiments, the cumulative totalproductivity for the cells is about any of the isoprene amountsdisclosed herein.

In some embodiments, the cells in culture produce isoprene at greaterthan or about 1, 10, 25, 50, 100, 150, 200, 250, 300, 400, 500, 600,700, 800, 900, 1,000, 1,250, 1,500, 1,750, 2,000, 2,500, 3,000, 4,000,5,000, 10,000, 12,500, 20,000, 30,000, 40,000, 50,000, 75,000, 100,000,125,000, 150,000, 188,000, or more nmole of isoprene/gram of cells forthe wet weight of the cells/hour (nmole/g_(wcm)/hr). In someembodiments, the amount of isoprene is between about 2 to about 200,000nmole/g_(wcm)/hr, such as between about 2 to about 100 nmole/g_(wcm)/hr,about 100 to about 500 nmole/g_(wcm)/hr, about 150 to about 500nmole/g_(wcm)/hr, about 500 to about 1,000 nmole/g_(wcm)/hr, about 1,000to about 2,000 nmole/g_(wcm)/hr, about 2,000 to about 5,000nmole/g_(wcm)/hr, about 5,000 to about 10,000 nmole/g_(wcm)/hr, about10,000 to about 50,000 nmole/g_(wcm)/hr, about 50,000 to about 100,000nmole/g_(wcm)/hr, about 100,000 to about 150,000 nmole/g_(wcm)/hr, orabout 150,000 to about 200,000 nmole/g_(wcm). In some embodiments, theamount of isoprene is between about 20 to about 200,000nmole/g_(wcm)/hr, about 100 to about 5,000 nmole/g_(wcm)/hr, about 200to about 2,000 nmole/g_(wcm)/hr, about 200 to about 1,000nmole/g_(wcm)/hr, about 300 to about 1,000 nmole/g_(wcm)/hr, about 400to about 1,000 nmole/g_(wcm)/hr, about 1,000 to about 5,000nmole/g_(wcm)/hr, about 2,000 to about 20,000 nmole/g_(wcm)/hr, about5,000 to about 50,000 nmole/g_(wcm)/hr, about 10,000 to about 100,000nmole/g_(wcm)/hr, about 20,000 to about 150,000 nmole/g_(wcm)/hr, orabout 20,000 to about 200,000 nmole/g_(wcm)/hr.

The amount of isoprene in units of nmole/g_(wcm)/hr can be measured asdisclosed in U.S. Pat. No. 5,849,970, which is hereby incorporated byreference in its entirety, particularly with respect to the measurementof isoprene production. For example, two mL of headspace (e.g.,headspace from a culture such as 2 mL of culture cultured in sealedvials at 32° C. with shaking at 200 rpm for approximately 3 hours) areanalyzed for isoprene using a standard gas chromatography system, suchas a system operated isothermally (85° C.) with an n-octane/porasil Ccolumn (Alltech Associates, Inc., Deerfield, Ill.) and coupled to a RGD2mercuric oxide reduction gas detector (Trace Analytical, Menlo Park,Calif.) (see, for example, Greenberg et al, Atmos. Environ. 27A:2689-2692, 1993; Silver et al., Plant Physiol. 97:1588-1591, 1991, whichare each hereby incorporated by reference in their entireties,particularly with respect to the measurement of isoprene production).The gas chromatography area units are converted to nmol isoprene via astandard isoprene concentration calibration curve. In some embodiments,the value for the grams of cells for the wet weight of the cells iscalculated by obtaining the A₆₀₀ value for a sample of the cell culture,and then converting the A₆₀₀ value to grams of cells based on acalibration curve of wet weights for cell cultures with a known A₆₀₀value. In some embodiments, the grams of the cells is estimated byassuming that one liter of broth (including cell medium and cells) withan A₆₀₀ value of 1 has a wet cell weight of 1 gram. The value is alsodivided by the number of hours the culture has been incubating for, suchas three hours.

In some embodiments, the cells in culture produce isoprene at greaterthan or about 1, 10, 25, 50, 100, 150, 200, 250, 300, 400, 500, 600,700, 800, 900, 1,000, 1,250, 1,500, 1,750, 2,000, 2,500, 3,000, 4,000,5,000, 10,000, 100,000, or more ng of isoprene/gram of cells for the wetweight of the cells/hr (ng/g_(wcm)/h). In some embodiments, the amountof isoprene is between about 2 to about 5,000 ng/g_(wcm)/h, such asbetween about 2 to about 100 ng/g_(wcm)/h, about 100 to about 500ng/g_(wcm)/h, about 500 to about 1,000 ng/g_(wcm)/h, about 1,000 toabout 2,000 ng/g_(wcm)/h, or about 2,000 to about 5,000 ng/g_(wcm)/h. Insome embodiments, the amount of isoprene is between about 20 to about5,000 ng/g_(wcm)/h, about 100 to about 5,000 ng/g_(wcm)/h, about 200 toabout 2,000 ng/g_(wcm)/h, about 200 to about 1,000 ng/g_(wcm)/h, about300 to about 1,000 ng/g_(wcm)/h, or about 400 to about 1,000ng/g_(wcm)/h. The amount of isoprene in ng/g_(wcm)/h can be calculatedby multiplying the value for isoprene production in the units ofnmole/g_(wcm)/hr discussed above by 68.1 (as described in Equation 5below).

In some embodiments, the cells in culture produce a cumulative titer(total amount) of isoprene at greater than or about 1, 10, 25, 50, 100,150, 200, 250, 300, 400, 500, 600, 700, 800, 900, 1,000, 1,250, 1,500,1,750, 2,000, 2,500, 3,000, 4,000, 5,000, 10,000, 50,000, 100,000, ormore mg of isoprene/L of broth (mg/L_(broth), wherein the volume ofbroth includes the volume of the cells and the cell medium). In someembodiments, the amount of isoprene is between about 2 to about 5,000mg/L_(broth), such as between about 2 to about 100 mg/L_(broth), about100 to about 500 mg/L_(broth), about 500 to about 1,000 mg/L_(broth),about 1,000 to about 2,000 mg/L_(broth), or about 2,000 to about 5,000mg/L_(broth). In some embodiments, the amount of isoprene is betweenabout 20 to about 5,000 mg/L_(broth), about 100 to about 5,000mg/L_(broth), about 200 to about 2,000 mg/L_(broth), about 200 to about1,000 mg/L_(broth), about 300 to about 1,000 mg/L_(broth), or about 400to about 1,000 mg/L_(broth).

The specific productivity of isoprene in mg of isoprene/L of headspacefrom shake flask or similar cultures can be measured by taking a 1 mlsample from the cell culture at an OD₆₀₀ value of approximately 1.0,putting it in a 20 mL vial, incubating for 30 minutes, and thenmeasuring the amount of isoprene in the headspace (as described, forexample, in Example I, part II). If the OD₆₀₀ value is not 1.0, then themeasurement can be normalized to an OD₆₀₀ value of 1.0 by dividing bythe OD₆₀₀ value. The value of mg isoprene/L headspace can be convertedto mg/L_(broth)/hr/OD₆₀₀ of culture broth by multiplying by a factor of38. The value in units of mg/L_(broth)/hr/OD₆₀₀ can be multiplied by thenumber of hours and the OD₆₀₀ value to obtain the cumulative titer inunits of mg of isoprene/L of broth.

In some embodiments, the cells in culture have an average volumetricproductivity of isoprene at greater than or about 0.1, 1.0, 10, 25, 50,100, 150, 200, 250, 300, 400, 500, 600, 700, 800, 900, 1,000, 1100,1200, 1300, 1,400, 1,500, 1,600, 1,700, 1,800, 1,900, 2,000, 2,100,2,200, 2,300, 2,400, 2,500, 2,600, 2,700, 2,800, 2,900, 3,000, 3,100,3,200, 3,300, 3,400, 3,500, or more mg of isoprene/L of broth/hr(mg/L_(broth)/hr, wherein the volume of broth includes the volume of thecells and the cell medium). In some embodiments, the average volumetricproductivity of isoprene is between about 0.1 to about 3,500mg/L_(broth)/hr, such as between about 0.1 to about 100 mg/L_(broth)/hr,about 100 to about 500 mg/L_(broth)/hr, about 500 to about 1,000mg/L_(broth)/hr, about 1,000 to about 1,500 mg/L_(broth)/hr, about 1,500to about 2,000 mg/L_(broth)/hrr, about 2,000 to about 2,500mg/L_(broth)/hr, about 2,500 to about 3,000 mg/L_(broth)/hr, or about3,000 to about 3,500 mg/L_(broth)/hr. In some embodiments, the averagevolumetric productivity of isoprene is between about 10 to about 3,500mg/L_(broth)/hr, about 100 to about 3,500 mg/L_(broth)/hr, about 200 toabout 1,000 mg/L_(broth)/hr, about 200 to about 1,500 mg/L_(broth)/hr,about 1,000 to about 3,000 mg/L_(broth)/hr, or about 1,500 to about3,000 mg/L_(broth)/hr.

In some embodiments, the cells culture have a peak volumetricproductivity of isoprene at greater than or about 0.5, 1.0, 10, 25, 50,100, 150, 200, 250, 300, 400, 500, 600, 700, 800, 900, 1,000, 1100,1200, 1300, 1,400, 1,500, 1,600, 1,700, 1,800, 1,900, 2,000, 2,100,2,200, 2,300, 2,400, 2,500, 2,600, 2,700, 2,800, 2,900, 3,000, 3,100,3,200, 3,300, 3,400, 3,500, 3,750, 4,000, 4,250, 4,500, 4,750, 5,000,5,250, 5,500, 5,750, 6,000, 6,250, 6,500, 6,750, 7,000, 7,250, 7,500,7,750, 8,000, 8,250, 8,500, 8,750, 9,000, 9,250, 9,500, 9,750, 10,000,12,500, 15,000, or more mg of isoprene/L of broth/hr (mg/L_(broth)/hr,wherein the volume of broth includes the volume of the cells and thecell medium). In some embodiments, the peak volumetric productivity ofisoprene is between about 0.5 to about 15,000 mg/L_(broth)/hr, such asbetween about 0.5 to about 10 mg/L_(broth)/hr, about 1.0 to about 100mg/L_(broth)/hr, about 100 to about 500 mg/L_(broth)/hr, about 500 toabout 1,000 mg/L_(broth)/hr, about 1,000 to about 1,500 mg/L_(broth)/hr,about 1,500 to about 2,000 mg/L_(broth)/hr, about 2,000 to about 2,500mg/L_(broth)/hr, about 2,500 to about 3,000 mg/L_(broth)/hr, about 3,000to about 3,500 mg/L_(broth)/hr, about 3,500 to about 5,000mg/L_(broth)/hr, about 5,000 to about 7,500 mg/L_(broth)/hr, about 7,500to about 10,000 mg/L_(broth)/hr, about 10,000 to about 12,500mg/L_(broth)/hr, or about 12,500 to about 15,000 mg/L_(broth)/hr. Insome embodiments, the peak volumetric productivity of isoprene isbetween about 10 to about 15,000 mg/L_(broth)/hr, about 100 to about2,500 mg/L_(broth)/hr, about 1,000 to about 5,000 mg/L_(broth)/hr, about2,500 to about 7,500 mg/L_(broth)/hr, about 5,000 to about 10,000mg/L_(broth)/hr, about 7,500 to about 12,500 mg/L_(broth)/hr, or about10,000 to about 15,000 mg/L_(broth)/hr.

The instantaneous isoprene production rate in mg/L_(broth)/hr/hr in afermentor can be measured by taking a sample of the fermentor off-gas,analyzing it for the amount of isoprene (in units such as mg of isopreneper L_(gas)) as described, for example, in Example I, part II andmultiplying this value by the rate at which off-gas is passed thougheach liter of broth (e.g., at 1 vvm (volume of air/volume ofbroth/minute) this is 60 L_(gas) per hour). Thus, an off-gas level of 1mg/L_(gas) corresponds to an instantaneous production rate of 60mg/L_(broth)/hr at air flow of 1 vvm. If desired, the value in the unitsmg/L_(broth)/hr can be divided by the OD₆₀₀ value to obtain the specificrate in units of mg/L_(broth)/hr/OD. The average value of mgisoprene/L_(gas) can be converted to the total product productivity(grams of isoprene per liter of fermentation broth, mg/L_(broth)) bymultiplying this average off-gas isoprene concentration by the totalamount of off-gas sparged per liter of fermentation broth during thefermentation. Thus, an average off-gas isoprene concentration of 0.5mg/L_(broth)/hr over 10 hours at 1 vvm corresponds to a total productconcentration of 300 mg isoprene/L_(broth).

In some embodiments, the cells in culture convert greater than or about0.0015, 0.002, 0.005, 0.01, 0.02, 0.05, 0.1, 0.12, 0.14, 0.16, 0.2, 0.3,0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.2, 1.4, 1.6, 2.0, 2.2, 2.4, 2.6,3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0, 11.0, 12.0, 13.0, 14.0, 15.0,16.0, 17.0, 18.0, 19.0, 20.0, 21.0, 22.0, 23.0, 23.2, 23.4, 23.6, 23.8,24.0, 25.0, 30.0, 31.0, 32.0, 33.0, 35.0, 37.5, 40.0, 45.0, 47.5, 50.0,55.0, 60.0, 65.0, 70.0, 75.0, 80.0, 85.0, or 90.0 molar % of the carbonin the cell culture medium into isoprene. In some embodiments, thepercent conversion of carbon into isoprene is between about 0.002 toabout 90.0 molar %, such as about 0.002 to about 0.005%, about 0.005 toabout 0.01%, about 0.01 to about 0.05%, about 0.05 to about 0.15%, 0.15to about 0.2%, about 0.2 to about 0.3%, about 0.3 to about 0.5%, about0.5 to about 0.8%, about 0.8 to about 1.0%, about 1.0 to about 1.6%,about 1.6 to about 3.0%, about 3.0 to about 5.0%, about 5.0 to about8.0%, about 8.0 to about 10.0%, about 10.0 to about 15.0%, about 15.0 toabout 20.0%, about 20.0 to about 25.0%, about 25.0% to 30.0%, about30.0% to 35.0%, about 35.0% to 40.0%, about 45.0% to 50.0%, about 50.0%to 55.0%, about 55.0% to 60.0%, about 60.0% to 65.0%, about 65.0% to70.0%, about 75.0% to 80.0%, about 80.0% to 85.0%, or about 85.0% to90.0%. In some embodiments, the percent conversion of carbon intoisoprene is between about 0.002 to about 0.4 molar %, 0.002 to about0.16 molar %, 0.04 to about 0.16 molar %, about 0.005 to about 0.3 molar%, about 0.01 to about 0.3 molar %, about 0.05 to about 0.3 molar %,about 0.1 to 0.3 molar %, about 0.3 to about 1.0 molar %, about 1.0 toabout 5.0 molar %, about 2 to about 5.0 molar %, about 5.0 to about 10.0molar %, about 7 to about 10.0 molar %, about 10.0 to about 20.0 molar%, about 12 to about 20.0 molar %, about 16 to about 20.0 molar %, about18 to about 20.0 molar %, about 18 to 23.2 molar %, about 18 to 23.6molar %, about 18 to about 23.8 molar %, about 18 to about 24.0 molar %,about 18 to about 25.0 molar %, about 20 to about 30.0 molar %, about 30to about 40.0 molar %, about 30 to about 50.0 molar %, about 30 to about60.0 molar %, about 30 to about 70.0 molar %, about 30 to about 80.0molar %, or about 30 to about 90.0 molar %.

The percent conversion of carbon into isoprene (also referred to as “%carbon yield”) can be measured by dividing the moles carbon in theisoprene produced by the moles carbon in the carbon source (such as themoles of carbon in batched and fed glucose and yeast extract). Thisnumber is multiplied by 100% to give a percentage value (as indicated inEquation 1).

$\begin{matrix}{{{Carbon}\mspace{14mu}{Yield}} = \frac{{moles}\mspace{14mu}{carbon}\mspace{14mu}{in}\mspace{14mu}{isoprene}\mspace{14mu}{produced}}{\left( {{moles}\mspace{14mu}{carbon}\mspace{14mu}{in}\mspace{14mu}{carbon}\mspace{14mu}{source}} \right) \times 100}} & {{Equation}\mspace{14mu} 1}\end{matrix}$

For this calculation, yeast extract can be assumed to contain 50% w/wcarbon. As an example, for the 500 liter described in Example 7, partVIII, the percent conversion of carbon into isoprene can be calculatedas shown in Equation 2.

$\begin{matrix}\begin{matrix}{\begin{matrix}{\%\mspace{14mu}{Carbon}} \\{Yield}\end{matrix} = \frac{\begin{matrix}{39.1\mspace{11mu} g\mspace{14mu}{isoprene} \times {1/}} \\{68.1\mspace{14mu}{mol}\text{/}g \times 5\mspace{14mu} C\text{/}{mol}}\end{matrix}}{\begin{matrix}\left\lbrack \left( {181,221\mspace{14mu} g\mspace{14mu}{glucose} \times {1/180}\mspace{14mu}{mol}\text{/}g \times} \right. \right. \\{\left. {6\mspace{14mu} C\text{/}{mol}} \right) + \left( {17,780\mspace{14mu} g\mspace{14mu}{yeast}\mspace{14mu}{extract} \times} \right.} \\{\left. \left. {0.5 \times {1/12}\mspace{14mu}{mol}\text{/}g} \right) \right\rbrack \times 100}\end{matrix}}} \\{= {0.042\;\%}}\end{matrix} & {{Equation}\mspace{14mu} 2}\end{matrix}$

For the two 500 liter fermentations described herein (Example 7, partsVII and VIII), the percent conversion of carbon into isoprene wasbetween 0.04-0.06%. A 0.11-0.16% carbon yield has been achieved using 14liter systems as described herein.

One skilled in the art can readily convert the rates of isopreneproduction or amount of isoprene produced into any other units.Exemplary equations are listed below for interconverting between units.

Units for Rate of Isoprene production (total and specific)1 g isoprene/L_(broth)/hr=14.7 mmol isoprene/L_(broth)/hr (totalvolumetric rate)  Equation 31 nmol isoprene/g_(wcm)/hr=1 nmol isoprene/L_(broth)/hr/OD₆₀₀ (Thisconversion assumes that one liter of broth with an OD₆₀₀ value of 1 hasa wet cell weight of 1 gram.)  Equation 41 nmol isoprene/g_(wcm)/hr=68.1 ng isoprene/g_(wcm)/hr (given themolecular weight of isoprene)  Equation 51 nmol isoprene/L_(gas) O₂/hr=90 nmol isoprene/L_(broth)/hr (at an O₂flow rate of 90 L/hr per L of culture broth)  Equation 61 ug isoprene/L_(gas) in off-gas=60 ug isoprene/L_(broth)/hr at a flowrate of 60 L_(gas) per L_(broth) (1 vvm)  Equation 7Units for Titer (Total and Specific)1 nmol isoprene/mg cell protein=150 nmol isoprene/L_(broth)/O₆₀₀ (Thisconversion assumes that one liter of broth with an OD₆₀₀ value of 1 hasa total cell protein of approximately 150 mg) (specificproductivity)  Equation 81 g isoprene/L_(broth)=14.7 mmol isoprene/L_(broth) (totaltiter)  Equation 9If desired, Equation 10 can be used to convert any of the units thatinclude the wet weight of the cells into the corresponding units thatinclude the dry weight of the cells.Dry weight of cells=(wet weight of cells)/3.3  Equation 10

In some embodiments encompassed by the invention, a cell comprising aheterologous nucleic acid encoding an isoprene synthase polypeptideproduces an amount of isoprene that is at least or about 2-fold, 3-fold,5-fold, 10-fold, 25-fold, 50-fold, 100-fold, 150-fold, 200-fold,400-fold, or greater than the amount of isoprene produced from acorresponding cell grown under essentially the same conditions withoutthe heterologous nucleic acid encoding the isoprene synthasepolypeptide.

In some embodiments encompassed by the invention, a cell comprising aheterologous nucleic acid encoding an isoprene synthase polypeptide andone or more heterologous nucleic acids encoding a DXS, IDI, and/or MVApathway polypeptide produces an amount of isoprene that is at least orabout 2-fold, 3-fold, 5-fold, 10-fold, 25-fold, 50-fold, 100-fold,150-fold, 200-fold, 400-fold, or greater than the amount of isopreneproduced from a corresponding cell grown under essentially the sameconditions without the heterologous nucleic acids.

Exemplary Isoprene Purification Methods

In some embodiments, any of the methods described herein further includerecovering the isoprene. For example, the isoprene produced using thecompositions and methods of the invention can be recovered usingstandard techniques. such as gas stripping, fractionation,adsorption/desorption, pervaporation, thermal or vacuum desorption ofisoprene from a solid phase, or extraction of isoprene immobilized orabsorbed to a solid phase with a solvent (see, for example, U.S. Pat.Nos. 4,703,007 and 4,570,029, which are each hereby incorporated byreference in their entireties, particularly with respect to isoprenerecovery and purification methods). In some embodiments, the recovery ofisoprene involves the isolation of isoprene in a liquid form (such as aneat solution of isoprene or a solution of isoprene in a solvent). Gasstripping involves the removal of isoprene vapor from the fermentationoff-gas stream in a continuous manner. Such removal can be achieved inseveral different ways including, but not limited to, adsorption to asolid phase, partition into a liquid phase, or direct condensation. Insome embodiments, membrane enrichment of a dilute isoprene vapor streamabove the dew point of the vapor resulting in the condensation of liquidisoprene.

The recovery of isoprene may involve one step or multiple steps. In someembodiments, the removal of isoprene vapor from the fermentation off-gasand the conversion of isoprene to a liquid phase are performedsimultaneously. For example, isoprene can be directly condensed from theoff-gas stream to form a liquid. In some embodiments, the removal ofisoprene vapor from the fermentation off-gas and the conversion ofisoprene to a liquid phase are performed sequentially. For example,isoprene may be adsorbed to a solid phase and then extracted from thesolid phase with a solvent.

In some embodiments, any of the methods described herein further includepurifying the isoprene. For example, the isoprene produced using thecompositions and methods of the invention can be purified using standardtechniques. Purification refers to a process through which isoprene isseparated from one or more components that are present when the isopreneis produced. In some embodiments, the isoprene is obtained as asubstantially pure liquid. Examples of purification methods include (i)distillation from a solution in a liquid extractant and (ii)chromatography. As used herein, “purified isoprene” means isoprene thathas been separated from one or more components that are present when theisoprene is produced. In some embodiments, the isoprene is at leastabout 20%, by weight, free from other components that are present whenthe isoprene is produced. In various embodiments, the isoprene is atleast or about 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90%, 95%, or 99%,by weight, pure. Purity can be assayed by any appropriate method, e.g.,by column chromatography, HPLC analysis, or GC-MS analysis.

In some embodiments, any of the methods described herein further includepolymerizing the isoprene. For example, standard methods can be used topolymerize the purified isoprene to form cis-polyisoprene or other downstream products using standard methods.

Additional methods and compositions are described in U.S. Provisionalpatent application No. 61/097,186, filed on Sep. 15, 2008 (now publishedas United States Patent Publication No. 2010/0086978 A1), U.S.Provisional patent application No. 61/097,189, filed on Sep. 15, 2008(now published as United States Patent Publication No. 2010/0184178 A1),U.S. Provisional patent application No. 61/097,163, filed on Sep. 15,2008, and U.S. patent application Ser. No. 12/335,071 (now published asUnited States Patent Publication No. 2009/0203102 A1) all of which areincorporated by reference in their entirties, particularly with respectto compositions and methods for producing isoprene.

The isoprene of this invention can be polymerized into useful polymers,including synthetic rubber, utilizing the same techniques that areapplicable to isoprene that is derived from petrochemical sources. Thepolymerization and recovery of such isoprene containing polymers aresuitably carried out according to various methods suitable for dienemonomer polymerization processes. This includes batchwise,semi-continuous, or continuous operations under conditions that excludeair and other atmospheric impurities, particularly oxygen and moisture.The polymerization of the isoprene monomer may also be carried out in anumber of different polymerization reactor systems, including but notlimited to bulk polymerization, vapor phase polymerization, solutionpolymerization, suspension polymerization, emulsion polymerization, andprecipitation polymerization systems. The commercially preferred methodsof polymerization are typically solution polymerization and emulsionpolymerization.

The polymerization reaction can also be initiated using a vast array ofdifferent polymerization initiators or catalyst systems. The initiatoror catalyst system used will be dependent upon the desiredcharacteristics of the isoprene containing polymer being synthesized.For instance, in cases where cis-1,4-polyisoprene rubber is being made aZiegler Natta catalyst system which is comprised of titaniumtetrachloride and triethyl aluminum can be utilized. In synthesizingother types of isoprene containing polymers other types of initiatorsystems may be needed. For instance, isoprene containing polymers can bemade using a free radical initiator, a redox initiator, an anionicinitiator, or a cationic initiator. The preferred initiation or catalystsystem will depend upon the polymer microstructure, molecular weight,molecular weight distribution, and chain branching desired. Thepreferred initiators will also depend upon whether the isoprene is beinghomopolymerized or copolymerized with additional monomers. In the caseof copolymers the initiator used will also depend upon whether it isdesirable for the polymer being made to have a random, non-random, ortapered distribution of repeat units that are derived of the particularmonomers. For instance, anionic initiators or controlled free radicalinitiators are typically used in synthesizing block copolymers havingisoprene blocks.

It is important for the initiator or catalyst system employed to becompatible with the type of polymerization system used. For instance, inemulsion polymerizations free radical initiators are typically utilized.In solution polymerizations anionic initiators, such as alkyl lithiumcompounds, are typically employed to initiate the polymerization. Anadvantage of free radical polymerization is that reactions can typicallybe carried out under less rigorous conditions than ionicpolymerizations. Free radical initiation systems also exhibit a greatertolerance of trace impurities.

Conventional emulsion recipes may also be employed in polymerizingisoprene in accordance with the present invention; however, somerestrictions and modifications may arise either from the inclusion ofadditional comonomers, or the restrictions on polymerization parameters.Ionic surfactants, known in the art, including sulfonate detergents andcarboxylate, sulfate, and phosphate soaps are useful in this invention.The level of ionic surfactant is computed based upon the total weight ofthe organic components and may range from about 2 to 30 parts by weightof ionic surfactant per 100 parts by weight of organic components.

Examples of free radical initiators that are useful in the practice ofthe present invention are those known as “redox” initiators, such ascombinations of chelated iron salts, sodium formaldehyde sulfoxylate,and organic hydroperoxides. Representative of organic hydroperoxides arecumene hydroperoxide, paramenthane hydroperoxide, and tertiary butylhydroperoxide. Tertiary butyl hydroperoxide (t-BHP), tertiary butylperacetate (t-BPA) and “azo” initiators, such as azobisiobutyronitrile(AIBN), are preferred. The reaction temperature utilized in free radicalpolymerizations is typically maintained in the range of 0° C. to 150° C.Temperatures between about 20° C. and 120° C. are generally preferredand temperatures within the range of 60° C. to 100° C. are normally mostpreferred. The reaction pressure is not critical. It is typically onlysufficiently high to maintain liquid phase reaction conditions; it maybe autogenic pressure, which will vary depending upon the components ofthe reaction mixture and the temperature, or it may be higher, e.g., upto 1000 psi.

In batch operations, the polymerization time can be varied as desiredfrom as little as a few minutes to as lone as several days.Polymerization in batch processes may be terminated when monomer is nolonger absorbed, or earlier, if desired, e.g., if the reaction mixturebecomes too viscous. In continuous operations, the polymerizationmixture may be passed through a reactor or series of reactors of anysuitable design. The polymerization reactions in such cases are suitablyadjusted by varying the residence time in the reactor system. Residencetimes vary with the type of reactor system and range from 10 to 15minutes to 24 or more hours. The concentration of monomer in thereaction mixture may vary upwards from 5 percent by weight of thereaction mixture, depending on the conditions employed; the range from20 to 80 percent by weight is preferred.

The polymerization of isoprene may also be carried out in a suitableorganic solvent that is liquid under the conditions of reaction andwhich is relatively inert. The solvent may have the same number ofcarbon atoms per molecule as the diene reactant or it may be in adifferent boiling range. Preferred organic solvents are normally alkanesand cycloalkanes.

The solvents can be comprised of one or more aromatic, paraffinic orcycloparaffinic compounds. These solvents will normally contain fromabout 4 carbon atoms per mole to about 10 carbon atoms per molecule andwill be liquid under the conditions of the polymerization. Somerepresentative examples of suitable organic solvents include pentane,isooctane, cyclohexane, methylcyclohexane, isohexane, n-heptane,n-octane, n-hexane, benzene, toluene, xylene, ethylbenzene,diethylbenzene, isobutylbenzene, petroleum ether, kerosene, petroleumspirits, petroleum naphtha, and the like, alone or in admixture.Aromatic hydrocarbons, such as benzene, toluene, isopropylbenzene,xylene, or halogenated aromatic compounds, such as chlorobenzene,bromobenzene, or orthodichlorobenzene, may also be employed, but are notpreferred in most cases. Other useful solvents include tetrahydrofuranand dioxane.

In the solution polymerization, there will normally be from 5 to 30weight percent monomers in the polymerization medium. Suchpolymerization media are, of course, comprised of the organic solventand monomers. In most cases, it will be preferred for the polymerizationmedium to contain from 10 to 25 weight percent monomers. It is generallymore preferred for the polymerization medium to contain 15 to 20 weightpercent monomers.

The polymerization is typically carried out to attain an essentiallycomplete conversion of monomers into polymer. Incremental monomeraddition, or a chain transfer agent, may be used in order to avoidexcessive gel formation. Such minor modifications are within the skillof the artisan. After the polymerization is complete, the polymer isrecovered from a slurry or solution of the polymer. A simple filtrationmay be adequate to separate polymer from diluent. Other means forseparating polymer from diluent may be employed. The polymer may betreated, separately or while slurried in the reaction mixture, in orderto separate residues. Such treatment may be with alcohols such asmethanol, ethanol, or isopropanol, with acidified alcohols, or withother similar polar liquids. In many cases the polymers are obtained inhydrocarbon solutions and the polymer can be recovered by coagulationwith acidified alcohol, e.g., rapidly stirred methanol or isopropanolcontaining 2% hydrochloric acid. Following this initial coagulation, thepolymers may be washed with an appropriate liquid, such as methanol.

As has been previously noted, the isoprene can also be copolymerizedwith one or more additional comonomers to make useful copolymers. Someadjustments in the polymerization recipe or reaction conditions may benecessary to obtain a satisfactory rate of polymer formation, dependingon the relative amount of isoprene included and the other monomersinvolved. Examples of comonomers that are useful in the practice of thisinvention include other diene monomers, such as 1,3-butadiene andhexadienes. Vinyl aromatic monomers can also be copolymerizable withisoprene to make useful polymers. Such vinyl aromatic monomers includestyrene, α-methylstyrene, divinyl benzene, vinyl chloride, vinylacetate, vinylidene chloride, methyl methacrylate, ethyl acrylate,vinylpyridine, acrylonitrile, methacrylonitrile, methacrylic acid,itaconic acid and acrylic acid. Mixtures of different comonomers canalso be employed at differing levels.

The isoprene monomer can also be copolymerized with one or moreadditional conjugated diolefin monomers. Those containing from 4 to 8carbon atoms are generally preferred for commercial purposes. Somespecific representative examples of conjugated diolefin monomers thatcan be copolymerized with isoprene include 1,3-butadiene,2,3-dimethyl-1,3-butadiene, piperylene, 3-butyl-1,3-octadiene,2-phenyl-1,3-butadiene, and the like, alone or in admixture.

Some representative examples of ethylenically unsaturated monomers thatcan copolymerized with isoprene include alkyl acrylates, such as methylacrylate, ethyl acrylate, butyl acrylate, methyl methacrylate and thelike; vinylidene monomers having one or more terminal CH₂═CH— groups;vinyl aromatics such as styrene, .alpha.-methylstyrene, bromostyrene,chlorostyrene, fluorostyrene and the like; α-olefins such as ethylene,propylene, 1-butene and the like; vinyl halides, such as vinylbromide,chloroethene (vinylchloride), vinylfluoride, vinyliodide,1,2-dibromoethene, 1,1-dichloroethene (vinylidene chloride),1,2-dichloroethene and the like; vinyl esters, such as vinyl acetate; α,β-olefinically unsaturated nitriles, such as acrylonitrile andmethacrylonitrile; α,β-olefinically unsaturated amides, such asacrylamide, N-methyl acrylamide, N,N-dimethylacrylamide, methacrylamideand the like. Functionalized monomers can also optionally becopolymerized with the isoprene in making useful rubbery polymers.Functionalized monomers of this type and methods by which they can beincorporated into rubbery polymers are described in U.S. Pat. Nos.6,627,721 and 6,936,669. The teachings of U.S. Pat. Nos. 6,627,721 and6,936,669 are incorporated herein by reference for the purpose ofdescribing such functionalized monomers and their incorporation intoisoprene containing polymers.

Rubbery polymers which are copolymers of one or more diene monomers withone or more other ethylenically unsaturated monomers will normallycontain from about 50 weight percent to about 99 weight percentconjugated diolefin monomers (including isoprene) and from about 1weight percent to about 50 weight percent of the other ethylenicallyunsaturated monomers in addition to the conjugated diolefin monomers.For example, rubbery copolymers of isoprene monomer with vinylaromaticmonomers, such as styrene-isoprene rubbers will normally which containfrom 50 to 95 weight percent isoprene and from 5 to 50 weight percentvinylaromatic monomers.

Vinyl aromatic monomers are probably the most important group ofethylenically unsaturated monomers which are commonly incorporated intoisoprene containing rubbers. Such vinyl aromatic monomers typicallycontain from 8 to 20 carbon atoms. Usually, the vinyl aromatic monomerwill contain from 8 to 14 carbon atoms. The most widely used vinylaromatic monomer is styrene. Some examples of vinyl aromatic monomersthat can be utilized include styrene, 1-vinylnaphthalene,2-vinylnaphthalene, α-methylstyrene, 4-phenylstyrene, 3-methylstyreneand the like.

Some representative examples of isoprene containing rubbery polymersinclude cis-1,3-polyisoprene homopolymer rubber, 3,4-polyisoprenerubber, styrene-isoprene rubber (SIR), β-methylstyrene-isoprene rubber,styrene-isoprene-butadiene rubber (SIBR), styrene-isoprene rubber (SIR),isoprene-butadiene rubber (IBR), α-methylstyrene-isoprene-butadienerubber and α-methylstyrene-styrene-isoprene-butadiene rubber. In caseswhere the rubbery polymer is comprised of repeat units that are derivedfrom two or more monomers, the repeat units which are derived from thedifferent monomers, including the isopren, will normally be distributedin an essentially random manner. The repeat units that are derived fromthe monomers differ from the monomer in that a double bond is normallyconsumed in by the polymerization reaction.

The rubbery polymer can be made by solution polymerization in a batchprocess by in a continuous process by continuously charging the isoprenemonomer and optionally additional monomers into a polymerization zone.The polymerization zone will typically be a polymerization reactor or aseries of polymerization reactors. The polymerization zone will normallyprovide agitation to keep the monomers, polymer, initiator, and modifierwell dispersed throughout the organic solvent the polymerization zone.Such continuous polymerizations are typically conducted in a multiplereactor system. The rubbery polymer synthesized is continuouslywithdrawn from the polymerization zone. The monomer conversion attainedin the polymerization zone will normally be at least about 85 percent.It is preferred for the monomer conversion to be at least about 90percent.

The polymerization can be initiated with an anionic initiator, such asan alkyl lithium compound. The alkyl lithium compounds that can be usedwill typically contain from 1 to about 8 carbon atoms, such as n-butyllithium. The amount of the lithium initiator utilized will vary with themonomers being polymerized and with the molecular weight that is desiredfor the polymer being synthesized. However, as a general rule, from 0.01to 1 phm (parts per 100 parts by weight of monomer) of the lithiuminitiator will be utilized. In most cases, from 0.01 to 0.1 phm of thelithium initiator will be utilized with it being preferred to utilize0.025 to 0.07 phm of the lithium initiator.

Such anionic polymerizations are optionally conducted in the presence ofpolar modifiers, such as alkyltetrahydrofurfuryl ethers. Somerepresentative examples of specific polar modifiers that can be usedinclude methyltetrahydrofurfuryl ether, ethyltetrahydrofurfuryl ether,propyltetrahydrofurfuryl ether, butyltetrahydrofurfuryl ether,hexyltetrahydrofurfuryl ether, octyltetrahydrofurfuryl ether,dodecyltetrahydrofurfuryl ether, diethyl ether, di-n-propyl ether,diisopropyl ether, di-n-butyl ether, tetrahydrofuran, dioxane, ethyleneglycol dimethyl ether, ethylene glycol diethyl ether, diethylene glycoldimethyl ether, diethylene glycol diethyl ether, triethylene glycoldimethyl ether, trimethylamine, triethylamine,N,N,N′,N′-tetramethylethylenediamine, N-methyl morpholine, N-ethylmorpholine, or N-phenyl morpholine.

The polar modifier will typically be employed at a level wherein themolar ratio of the polar modifier to the lithium initiator is within therange of about 0.01:1 to about 5:1. The molar ratio of the polarmodifier to the lithium initiator will more typically be within therange of about 0.1:1 to about 4:1. It is generally preferred for themolar ratio of polar modifier to the lithium initiator to be within therange of about 0.25:1 to about 3:1. It is generally most preferred forthe molar ratio of polar modifier to the lithium initiator to be withinthe range of about 0.5:1 to about 3:2.

The polymerization temperature utilized in such anionic polymerizationscan vary over a broad range of from about −20° C. to about 180° C. Inmost cases, a polymerization temperature within the range of about 30°C. to about 125° C. will be utilized. It is typically preferred for thepolymerization temperature to be within the range of about 45° C. toabout 100° C. It is typically most preferred for the polymerizationtemperature to be within the range of about 60° C. to about 90° C. Thepressure used will normally be sufficient to maintain a substantiallyliquid phase under the conditions of the polymerization reaction.

Such anionic polymerizations of isoprene are normally conducted for alength of time sufficient to permit substantially completepolymerization of the isoprene and any additional monomers that arepresent. In other words, the polymerization is normally carried outuntil high conversions of at least about 85 percent are attained. Thepolymerization is then normally terminated by the addition of an agent,such as an alcohol, a terminating agent, or a coupling agent. Forexample, a tin halide and/or silicon halide can be used as a couplingagent. The tin halide and/or the silicon halide are continuously addedin cases where asymmetrical coupling is desired. This continuousaddition of tin coupling agent and/or the silicon coupling agent isnormally done in a reaction zone separate from the zone where the bulkof the polymerization is occurring. The coupling agents will normally beadded in a separate reaction vessel after the desired degree ofconversion has been attained. The coupling agents can be added in ahydrocarbon solution, e.g., in cyclohexane, to the polymerizationadmixture with suitable mixing for distribution and reaction. In otherwords, the coupling will typically be added only after a high degree ofconversion has already been attained. For instance, the coupling agentwill normally be added only after a monomer conversion of greater thanabout 85 percent has been realized. It will typically be preferred forthe monomer conversion to reach at least about 90 percent before thecoupling agent is added.

The tin halides used as coupling agents will normally be tintetrahalides, such as tin tetrachloride, tin tetrabromide, tintetrafluoride or tin tetraiodide. However, tin trihalides can alsooptionally be used. Polymers coupled with tin trihalides having amaximum of three arms. This is, of course, in contrast to polymerscoupled with tin tetrahalides which have a maximum of four arms. Toinduce a higher level of branching, tin tetrahalides are normallypreferred. As a general rule, tin tetrachloride is most preferred.

The silicon coupling agents that can be used will normally be silicontetrahalides, such as silicon tetrachloride, silicon tetrabromide,silicon tetrafluoride or silicon tetraiodide. However, silicontrihalides can also optionally be used. Polymers coupled with silicontrihalides having a maximum of three arms. This is, of course, incontrast to polymers coupled with silicon tetrahalides which have amaximum of four arms. To induce a higher level of branching, silicontetrahalides are normally preferred. As a general rule, silicontetrachloride is most preferred of the silicon coupling agents.

A combination of a tin halide and a silicon halide can optionally beused to couple the rubbery polymer. By using such a combination of tinand silicon coupling agents improved properties for tire rubbers, suchas lower hysteresis, can be attained. It is particularly desirable toutilize a combination of tin and silicon coupling agents in tire treadcompounds that contain both silica and carbon black. In such cases, themolar ratio of the tin halide to the silicon halide employed in couplingthe rubbery polymer will normally be within the range, of 20:80 to 95:5.The molar ratio of the tin halide to the silicon halide employed incoupling the rubbery polymer will more typically be within the range of40:60 to 90:10. The molar ratio of the tin halide to the silicon halideemployed in coupling the rubbery polymer will preferably be within therange of 60:40 to 85:15. The molar ratio of the tin halide to thesilicon halide employed in coupling the rubbery polymer will mostpreferably be within the range of 65:35 to 80:20.

Broadly, and exemplary, a range of about 0.01 to 4.5 milliequivalents oftin coupling agent (tin halide and silicon halide) is employed per 100grams of the rubbery polymer. It is normally preferred to utilize about0.01 to about 1.5 milliequivalents of the coupling agent per 100 gramsof polymer to obtain the desired Mooney viscosity. The larger quantitiestend to result in production of polymers containing terminally reactivegroups or insufficient coupling. One equivalent of tin coupling agentper equivalent of lithium is considered an optimum amount for maximumbranching. For instance, if a mixture tin tetrahalide and silicontetrahalide is used as the coupling agent, one mole of the couplingagent would be utilized per four moles of live lithium ends. In caseswhere a mixture of tin trihalide and silicon trihalide is used as thecoupling agent, one mole of the coupling agent will optimally beutilized for every three moles of live lithium ends. The coupling agentcan be added in a hydrocarbon solution, e.g., in cyclohexane, to thepolymerization admixture in the reactor with suitable mixing fordistribution and reaction.

After the coupling has been completed, a tertiary chelating alkyl1,2-ethylene diamine or a metal salt of a cyclic alcohol can optionallybe added to the polymer cement to stabilize the coupled rubbery polymer.In most cases, from about 0.01 phr (parts by weight per 100 parts byweight of dry rubber) to about 2 phr of the chelating alkyl 1,2-ethylenediamine or metal salt of the cyclic alcohol will be added to the polymercement to stabilize the rubbery polymer. Typically, from about 0.05 phrto about 1 phr of the chelating alkyl 1,2-ethylene diamine or metal saltof the cyclic alcohol will be added. More typically, from about 0.1 phrto about 0.6 phr of the chelating alkyl 1,2-ethylene diamine or themetal salt of the cyclic alcohol will be added to the polymer cement tostabilize the rubbery polymer.

The terminating agents that can be used to stop the polymerization andto “terminate” the living rubbery polymer include tin monohalides,silicon monohalides, N,N,N′,N′-tetradialkyldiamino-benzophenones (suchas tetramethyldiaminobenzophenone and the like),N,N-dialkylamino-benzaldehydes (such as dimethylaminobenzaldehyde andthe like), 1,3-dialkyl-2-imidazolidinones (such as1,3-dimethyl-2-imidazolidinone and the like), 1-alkyl substitutedpyrrolidinones; 1-aryl substituted pyrrolidinones,dialkyl-dicycloalkyl-carbodiimides containing from about 5 to about 20carbon atoms, and dicycloalkyl-carbodiimides containing from about 5 toabout 20 carbon atoms.

After the termination step, and optionally the stabilization step, hasbeen completed, the rubbery polymer can be recovered from the organicsolvent. The coupled rubbery polymer can be recovered from the organicsolvent and residue by means such as chemical (alcohol) coagulation,thermal desolventization, or other suitable method. For instance, it isoften desirable to precipitate the rubbery polymer from the organicsolvent by the addition of lower alcohols containing from about 1 toabout 4 carbon atoms to the polymer solution. Suitable lower alcoholsfor precipitation of the rubber from the polymer cement includemethanol, ethanol, isopropyl alcohol, normal-propyl alcohol and t-butylalcohol. The utilization of lower alcohols to precipitate the rubberypolymer from the polymer cement also “terminates” any remaining livingpolymer by inactivating lithium end groups. After the coupled rubberypolymer is recovered from the solution, steam-stripping can be employedto reduce the level of volatile organic compounds in the coupled rubberypolymer. Additionally, the organic solvent can be removed from therubbery polymer by drum drying, extruder drying, vacuum drying, and thelike.

As has previously been explained, synthetic cis-1,3-polyisoprene rubberthat is similar enough to allow for free substitution with naturalrubber can be produced by the solution polymerization of isoprene with aZiegler Natta catalyst system that is comprised of titaniumtetrachloride (TiCl₄) and an organoaluminum compound, such as triethylaluminum, Al—(CH₂—CH₃)₃. The polyisoprene rubber that is made with thisZiegler Natta catalyst system has a high cis-microstructure contain ofup to 98 percent that closely assimilates that of natural rubber fromHevea Brasiliensis (the common rubber tree) which has acic-microstructure content of virtually 100 percent. However, thisslight difference in polymer microstructure results of physicalproperties that are inferior to those of natural rubber is certainrespects. For instance, natural rubber typically exhibits green strengththat is superior to that of synthetic cis-1,4-polyisprene rubber. On theother hand, in certain other respects synthetic cis-1,4-polyisprenerubber is superior to natural rubber from the Hevea Brasiliensis,guayule, and Taraxacum kok-Saghyz (Russian dandelion). For instance,natural rubber contains residual proteins, soaps, resins, and sugarssince it comes from plants. The presence of these residual impuritiescan be extremely detrimental in some applications. For instance, thepresence of residual proteins in rubber products can cause seriousallergic reactions in some people and are a mojor concern formanufacturers of some rubber-containing products, such as rubber gloves,condoms, syringe plungers, and the like. In any case, the syntheticpolyisoprene homopolymer rubbers of this invention that are free fromproteins, soaps, resins, and sugars present in natural rubber, includingnatural rubber from the Hevea Brasiliensis.

U.S. Pat. No. 3,931,136 discloses a process for producing high molecularweight cis-1,4-polyisoprene. The catalyst used in this process is athree-component mixture of (A) a titanium tetrachloride, (B) anorganoaluminum compound of the formula AlR₃, where each R represents analkyl group, preferably an alkyl group containing 1 to 8 carbon atoms,an aryl group, preferably a phenyl group, or a cycloalkyl group,preferably a cyclohexyl group, and (C) a beta-diketone of the formula:

where R′ and R″ can be the same or different and represent an alkylgroup or a aryl group. R′ and R″ will preferably represent an alkylgroup containing from 1 to 5 carbon atoms or a phenyl group. Theteachings of U.S. Pat. No. 3,931,136 are incorporated herein byreference for the purpose of teaching catalyst systems andpolymerization techniques that can be used in synthesizingcis-1,4-polyisoprene.

A solution polymerization technique for synthesizingcis-1,4-polyisoprene with a catalyst system that is comprised of amixture of titanium tetrachloride and a trialkylaluminum compound isdisclosed by U.S. Pat. No. 4,430,487. In this process the polymerizationis shortstopped with 4,7-diaza-decane-1,10-diamine. The teachings ofU.S. Pat. No. 4,430,487 are incorporated herein by reference for thepurpose of teaching catalyst systems and polymerization techniques thatcan be used in synthesizing cis-1,4-polyisoprene.

The synthesis of cis-1,4-polyisoprene by polymerizing isoprene with acatalyst system which is comprised of a titanium tetrahalide, atrialkylaluminum compound and diphenylether can result in the formationof unwanted gel. U.S. Pat. No. 5,919,876 discloses that gel formationcan be reduced by conducting such polymerizations in the presence of adiarylamine, such as para-styrenated diphenylamine. U.S. Pat. No.5,919,876 more specifically discloses a process for synthesizingcis-1,4-polyisoprene having a low gel content which comprisespolymerizing isoprene in an inert organic solvent with a preformedcatalyst system which is made by reacting an organoaluminum compoundwith titanium tetrahalide, such as titanium tetrachloride, in thepresence of at least one ether, wherein said polymerization is conductedat a temperature which is within the range of about 0° C. to about 100°C., and wherein said polymerization is conducted in the presence of adiarylamine. The teachings of U.S. Pat. No. 5,919,867 are incorporatedherein by reference for the purpose of teaching catalyst systems andsolution polymerization techniques that can be used in synthesizingcis-1,4-polyisoprene rubber.

Cis-1,4-polyisoprene can be made by vapor phase polymerization utilizinga preformed catalyst that is made by reacting an organoaluminum compoundwith titanium tetrachloride. U.S. Pat. No. 6,066,705 discloses a methodfor vapor phase polymerizing isoprene into cis-1,4-polyisoprene in aprocess comprising the steps of: (1) charging into a reaction zone saidisoprene and a preformed catalyst system which is made by reacting anorganoaluminum compound with titanium tetrachloride, preferably in thepresence of at least one ether; wherein the isoprene is maintained inthe vapor phase in said reaction zone by a suitable combination oftemperature and pressure; (2) allowing said isoprene to polymerize intocis-1,4-polyisoprene at a temperature within the range of about 35° C.to about 70° C.; and (3) withdrawing said cis-1,4-polyisoprene from saidreaction zone. It has been determined that gel formation can be reducedin such vapor phase polymerizations by conducting the polymerization ofthe isoprene monomer in the presence of a diarylamine, such aspara-styrenated diphenylamine. The teachings of U.S. Pat. No. 6,066,705are incorporated herein by reference for the purpose of teachingcatalyst systems and vapor phase polymerization techniques that can beused in synthesizing cis-1,4-polyisoprene rubber.

Polyisoprene rubber that is clear (transparent) and of high purity canbe synthesized utilizing a neodymium catalyst system. U.S. Pat. No.6,780,948 relates to such a process for the synthesis of polyisoprenerubber which comprises polymerizing isoprene monomer in the presence ofa neodymium catalyst system, wherein the neodymium catalyst system isprepared by (1) reacting a neodymium carboxylate with an organoaluminumcompound in the presence of isoprene for a period of about 10 minutes toabout 30 minutes to produce neodymium-aluminum catalyst component, and(2) subsequently reacting the neodymium-aluminum catalyst component witha dialkyl aluminum chloride for a period of at least 30 minutes toproduce the neodymium catalyst system. The teachings of U.S. Pat. No.5,919,867 are incorporated herein by reference for the purpose ofteaching catalyst systems and polymerization techniques that can be usedin synthesizing cis-1,4-polyisoprene rubber that is of high purity.

U.S. Pat. Nos. 7,091,150 and 7,199,201 disclose the use of a neodymiumcatalyst system to polymerize isoprene monomer into syntheticpolyisoprene rubber having an extremely high cis-microstructure contentand high stereo regularity. This polyisoprene rubber will crystallizeunder strain and can be compounded into rubber formulations in a mannersimilar to natural rubber. This technique more specifically discloses aprocess for the synthesis of polyisoprene rubber which comprisespolymerizing isoprene monomer in the presence of a neodymium catalystsystem, wherein the neodymium catalyst system is prepared by a processthat comprises (1) reacting a neodymium carboxylate with anorganoaluminum compound in an organic solvent to produceneodymium-aluminum catalyst component, and (2) subsequently reacting theneodymium-aluminum catalyst component with an elemental halogen toproduce the neodymium catalyst system. In practicing this process, theneodymium catalyst system is typically void of nickel-containingcompounds.

The synthetic polyisoprene rubber made by this process is comprised ofrepeat units that are derived from isoprene, wherein the syntheticpolyisoprene rubber has a cis-microstructure content which is within therange of 98.0% to 99.5%, a 3,4-microstructure content which is withinthe range of 0.5% to 2.0%, and a trans-microstructure content which iswithin the range of 0.0% to 0.5%. The teachings of U.S. Pat. Nos.7,091,150 and 7,199,201 are incorporated herein by reference for thepurpose of teaching neodymium catalyst systems and polymerizationtechniques that can be used in synthesizing cis-1,4-polyisoprene rubberof extremely high cis-microstructure content and high stereo regularity.

Single component lanthanide catalysts, such as lanthanide diiodides, canalso be used in the synthesis of polyisoprene having extremely highcis-microstructure contents. For instance, thulium diiodide, dysprosiumdiiodide, and neodymium diiodide can initiate the polymerization ofisoprene into high cis-1,4-polyisoprene rubber without the need for anyadditional catalyst components. Lanthanide diiodides can accordingly beused to initiate the polymerization of isoprene monomer into highcis-1,4-polyisoprene under solution polymerization conditions.

U.S. Pat. No. 4,894,425 reveals a process for synthesizing polyisoprenethat may possess functional groups and that contains more than 70percent 1,2- and 3,4-structural units. This process involves the anionicpolymerization of isoprene in an inert hydrocarbon solvent in thepresence of an organolithium compound as the catalyst and an ether asthe cocatalyst, wherein the cocatalyst used is an ethylene glycoldialkyl ether of the formula R¹—O—CH₂—CH₂—O—R² wherein R¹ and R² arealkyl groups having different numbers of carbon atoms, selected from thegroup consisting of methyl, ethyl, n-propyl, iso-propyl, n-butyl,iso-butyl, sec-butyl, and tert-butyl, and wherein the sum of the carbonatoms in the two alkyl groups R¹ and R² is within the range of 5 to 7.The teachings of U.S. Pat. No. 4,894,425 are incorporated herein byreference for the purpose of teaching catalyst systems andpolymerization techniques that can be used in synthesizing polyisoprenehaving a high 1,2- and 3,4-microstructure content.

Crystallizable 3,4-polyisoprene can be synthesized in organic solventsto quantitative yields after short polymerization times by utilizing thecatalyst systems described by U.S. Pat. No. 5,082,906. The3,4-polyisoprene made utilizing this catalyst system is straincrystallizable and can be employed in tire treads which provide improvedtraction and improved cut growth resistance. U.S. Pat. No. 5,082,906specifically discloses a process for the synthesis of 3,4-polyisoprenewhich comprises polymerizing isoprene monomer in an organic solvent at atemperature which is within the range of about −10° C. to about 100° C.in the presence of a catalyst system which is composed of (a) anorganoiron compound, (b) an organoaluminum compound, (c) a chelatingaromatic amine, and (d) a protonic compound; wherein the molar ratio ofthe chelating amine to the organoiron compound is within the range ofabout 0.1:1 to about 1:1, wherein the molar ratio of the organoaluminumcompound to the organoiron compound is within the range of about 5:1 toabout 200:1, and wherein the molar ratio of the protonic compound to theorganoaluminum compound is within the range of about 0.001:1 to about0.2:1. The teachings of U.S. Pat. No. 5,082,906 are incorporated hereinby reference for the purpose of teaching catalyst systems andpolymerization techniques that can be used in synthesizing polyisoprenehaving a high 3,4-microstructure content and which is straincrystallizable.

U.S. Pat. No. 5,356,997 also relates to a process for the synthesis ofstrain crystallizable 3,4-polyisoprene. This 3,4-polyisoprene has a3,4-microstructure content which is within the range of about 65% toabout 85%, a cis-1,4-microstructure content which is within the range ofabout 15% to about 35%, and essentially no trans-1,4-microstructure or1,2-microstructure. It can be synthesized in organic solvents toquantitative yields after short polymerization times. U.S. Pat. No.5,356,997 specifically discloses a process for the synthesis of3,4-polyisoprene which comprises polymerizing isoprene monomer in anorganic solvent at a temperature which is within the range of about −10°C. to about 100° C. in the presence of a catalyst system which iscomprised of (a) an organoiron compound which is soluble in the organicsolvent, wherein the iron in the organoiron compound is in the +3oxidation state, (b) a partially hydrolyzed organoaluminum compoundwhich was prepared by adding a protonic compound selected from the groupconsisting of water, alcohols and carboxylic acids to the organoaluminumcompound, and (c) a chelating aromatic amine; wherein the molar ratio ofthe chelating amine to the organoiron compound is within the range ofabout 0.1:1 to about 1:1, wherein the molar ratio of the organoaluminumcompound to the organoiron compound is within the range of about 5:1 toabout 200:1, and wherein the molar ratio of the protonic compound to theorganoaluminum compound is within the range of about 0.001:1 to about0.2:1. The teachings of U.S. Pat. No. 5,356,997 are incorporated hereinby reference for the purpose of teaching catalyst systems andpolymerization techniques that can be used in synthesizing polyisoprenehaving a high 3,4-microstructure content and which is straincrystallizable.

U.S. Pat. No. 5,677,402 reveals a process for preparing 3,4-polyisoprenerubber which comprises polymerizing isoprene monomer with anorganolithium initiator at a temperature which is within the range ofabout 30° C. to about 100° C. in the presence of a sodium alkoxide and apolar modifier, wherein the molar ratio of the sodium alkoxide to theorganolithium initiator is within the range of about 0.05:1 to about3:1; and wherein the molar ratio of the polar modifier to theorganolithium initiator is within the range of about 0.25:1 to about5:1. The teachings of U.S. Pat. No. 5,677,402 are incorporated herein byreference for the purpose of teaching catalyst systems andpolymerization techniques that can be used in synthesizing3,4-polyisoprene.

U.S. Pat. No. 7,351,768 discloses the synthesis of liquid polyisoprenehaving a weight average molecular weight which is within the range of5,000 to 100,000 and preferable within the range of 20,000 to 80,000.The teachings of U.S. Pat. No. 5,677,402 are incorporated herein byreference for the purpose illustrating the synthesis of liquidpolyisoprene.

U.S. Pat. No. 6,576,728 discloses a process for the copolymerization ofstyrene and isoprene to produce low vinyl styrene-isoprene rubber havinga random distribution of repeat units that are derived from styrene. Theinitiator systems employed in these polymerizations are comprised of (a)a lithium initiator and (b) a member selected from the group consistingof (1) a sodium alkoxide, (2) a sodium salt of a sulfonic acid, and (3)a sodium salt of a glycol ether. It is important for the initiatorsystem used in these polymerzations to be free of polar modifiers, suchas Lewis bases. The teachings of U.S. Pat. No. 6,576,728 areincorporated herein by reference for the purpose illustrating thesynthesis of styrene-isoprene rubber.

U.S. Pat. No. 6,313,216 discloses a process for synthesizing randomstyrene-isoprene rubber comprising: (1) continuously charging isoprene,styrene, an initiator, and a solvent into a first polymerization zone,(2) allowing the isoprene and styrene to copolymerize in the firstpolymerization zone to total conversion of 60 to 95 percent to produce apolymer cement containing living styrene-isoprene chains, (3)continuously charging the polymer cement containing livingstyrene-isoprene chains and additional isoprene monomer into a secondpolymerization zone, wherein from 5 to 40 percent of the total amount ofisoprene changed is charged into the second polymerization zone, (4)allowing the copolymerization to continue in the second polymerizationzone to a conversion of the isoprene monomer of at least 90 percentwherein the total conversion of styrene and isoprene in the secondpolymerization zone is limited to a maximum of 98 percent, (5)withdrawing a polymer cement of random styrene-isoprene rubber havingliving chain ends from the second reaction zone, (6) killing the livingchain ends on the random styrene-isoprene rubber, and (7) recovering therandom styrene-isoprene rubber from the polymer cement, wherein thecopolymerizations in the first polymerization zone and the secondpolymerization zone are carried out at a temperature which is within therange of 70° C. to 100° C., and wherein the amount of styrene chargedinto the first polymerization zone is at least 2 percent more than thetotal amount of styrene bound into the rubber. The teachings of U.S.Pat. No. 6,313,216 are incorporated herein by reference for the purposeillustrating the synthesis of styrene-isoprene rubber.

Isoprene-butadiene copolymers having high vinyl contents can besynthesized in organic solvents to high yields after shortpolymerization times by utilizing the process disclosed in U.S. Pat. No.5,061,765. The isoprene-butadiene copolymers made utilizing this processhave a glass transition temperature which is within the range of about0° C. to about −60° C. and can be employed in tire treads which provideimproved traction and improved cut growth resistance. U.S. Pat. No.5,061,765 more specifically discloses a process for the synthesis ofisoprene-butadiene copolymers having a high vinyl content whichcomprises copolymerizing isoprene monomer and butadiene monomer in anorganic solvent at a temperature which is within the range of about −10°C. to about 100° C. in the presence of a catalyst system which iscomprised of (a) an organoiron compound, (b) an organoaluminum compound,(c) a chelating aromatic amine, and (d) a protonic compound; wherein themolar ratio of the chelating amine to the organoiron compound is withinthe range of about 0.1:1 to about 1:1, wherein the molar ratio of theorganoaluminum compound to the organoiron compound is within the rangeof about 5:1 to about 200:1, and wherein the molar ratio of the protoniccompound to the organoaluminum compound is within the range of about0.001:1 to about 0.2:1. The teachings of U.S. Pat. No. 5,061,765 areincorporated herein by reference for the purpose illustrating thesynthesis of isoprene-butadiene rubber.

A technique for synthesizing rubbery terpolymers of styrene, isopreneand butadiene is disclosed in U.S. Pat. No. 5,137,998. These rubberyterpolymers exhibit an excellent combination of properties forutilization in tire tread rubber compounds. By utilizing suchterpolymers in tire treads, tires having improved wet skid resistancecan be built without sacrificing rolling resistance or tread wearcharacteristics. U.S. Pat. No. 5,137,998 more specifically discloses aprocess for preparing a rubbery terpolymer of styrene, isoprene, andbutadiene having multiple glass transition temperatures and having anexcellent combination of properties for use in making tire treads whichcomprises: terpolymerizing styrene, isoprene and 1,3-butadiene in anorganic solvent at a temperature of no more than about 40° C. in thepresence of (a) at least one member selected from the group consistingof tripiperidino phosphine oxide and alkali metal alkoxides and (b) anorganolithium compound. The teachings of U.S. Pat. No. 5,137,998 areincorporated herein by reference for the purpose illustrating thesynthesis of styrene-isoprene-butadiene rubber.

A liquid isoprene-butadiene rubber (IBR) which is particularly valuablefor use in making treads for high performance automobile tires,including race tires, that exhibit superior dry traction characteristicsand durability, can be made by the process disclosed in U.S. Pat. No.6,562,895. This isoprene-butadiene rubber is a liquid at roomtemperature and is comprised of repeat units which are derived fromabout 5 weight percent to about 95 weight percent isoprene and fromabout 5 weight percent to about 95 weight percent 1,3-butadiene, whereinthe repeat units derived from isoprene and 1,3-butadiene are inessentially random order. This IBR also has a low number averagemolecular weight which is within the range of about 3,000 to about50,000 and has a glass transition temperature which is within the rangeof about −50° C. to about 20° C.

These isoprene-butadiene copolymers are synthesized utilizing anorganolithium initiator and a polar modifier. The level of organolithiuminitiator employed will be dependent upon the molecular weight which isdesired for the liquid isoprene-butadiene polymer being synthesized. Asa general rule, in all anionic polymerizations the molecular weight ofthe polymer produced is inversely proportional to the amount ofinitiator utilized. Since liquid isoprene-butadiene polymer having arelatively low molecular weight is being synthesized, the amount ofinitiator employed will be relatively large. As a general rule, fromabout 0.1 to about 2 phm (parts per hundred parts of monomer by weight)of the organolithium compound will be employed. In most cases, it willbe preferred to utilize from about 0.2 to about 1 phm of theorganolithium compound with it being most preferred to utilize fromabout 0.4 phm to 0.6 phm of the organolithium compound. In any case, anamount of organolithium initiator will be selected to result in theproduction of liquid isoprene-butadiene polymer having a number averagemolecular weight which is within the range of about 3,000 to about50,000.

The amount of organolithium initiator will preferably be selected toresult in the production of liquid isoprene-butadiene polymer having anumber average molecular weight which is within the range of about 5,000to about 30,000. The amount of organolithium initiator will mostpreferably be selected to result in the production of liquidisoprene-butadiene polymer having a number average molecular weight thatis within the range of about 8,000 to about 18,000. In any case, it iscritical to carry out the copolymerization of the 1,3-butadiene and thestyrene in the presence of a polar modifier, such asN,N,N′,N′-tetramethylethylenediamine (TMEDA), to attain a high glasstransition temperature which is within the range of about −50° C. to 20°C. The teachings of U.S. Pat. No. 6,562,895 are incorporated herein byreference for the purpose illustrating the synthesis of liquidisoprene-butadiene polymers.

Block copolymers containing a block of polyisoprene can be made by theprocess described in U.S. Pat. No. 5,242,984. For instance, lineardiblock polymers of styrene and isoprene (S-I block copolymers) andlinear triblock polymers of styrene and isoprene (S-I-S triblockpolymers) can be made by this process. In this technique, the monomersare polymerized sequentially by anionic polymerization in an inertorganic solvent. Normally an organoalkali metal compound, such as analkyl lithium compound, is used to initiate the polymerization which canbe conducted over a broad temperature range.

Methods of controlling the molecular weights of the blocks and theoverall polymer are described in U.S. Pat. Nos. 3,149,182 and 3,231,635which state that the amount of monomer can be kept constant anddifferent molecular weights can be achieved by changing the amount ofcatalyst or that the amount of catalyst can be kept constant anddifferent molecular weights can be achieved by varying the amount of themonomer. Following the sequential polymerization, the product isterminated such as by the addition of a protic terminating agent, e.g.water, alcohol or other reagents or with hydrogen, for the purpose ofremoving the lithium radical forming the nucleus for the condensedpolymer product. The block polymer product is then recovered such as bycoagulation utilizing hot water or steam or both. The teachings of U.S.Pat. Nos. 5,242,984, 3,149,182, and 3,231,635 are incorporated herein byreference for the purpose of teaching methods for synthesizing S-I blockcopolymers and S-I-S triblock polymers.

All types of polymers made with the isoprene of this invention areverifiable as being made with isoprene that did not originate from apetrochemical source. Additionally, the isoprene containing polymers ofthis invention can also be distinguished from isoprene containingpolymers that come from natural sources, such as natural rubber.Accordingly, the isoprene containing polymers of this invention areanalytically verifiable as coming from the bio-renewable,environmentally friendly, sources delineated herein.

The ratio of carbon isotopes ¹³C and ¹²C can be used to identify or ruleout potential origins for many carbon-containing samples. This methodworks well because: (1) both isotopes are stable on geological timeframes; (2) the ratio of ¹³C to ¹²C can be measured with great precisionusing combinations of combustion analysis, gas chromatography, andisotope ratio mass spectrometry; (3) ¹³C/¹²C ratios for many naturallyoccurring materials occur within narrow ranges characteristic of thosematerials; and (4) ¹³C/¹²C ratios for many materials change inpredictable ways as these materials undergo chemical reactions.

Studies involving ¹³C/¹²C ratios at or near natural abundance levelsusually report isotopic data as “delta values”, which are represented bythe symbol δ¹³C and given in parts per thousand (‰) relative to astandard reference sample. For carbon, the reference sample typically isPee Dee Belemite, which has a ¹³C natural abundance of 1.112328% and isassigned δ¹³C 0.00‰. The formula relating ¹³C/¹²C ratios to delta valuesis:δ¹³C (in ‰) versus standard=[(R _(sample) −R _(standard))R_(standard)](1000), where R _(sample) is the ¹³C/¹²C ration for thesample and R_(standard) is the ratio for Pee Dee Belemite.

Although isotopes of carbon (i.e., ¹³C and ¹²C) take part in the samephysical processes and same chemical reactions, the slight massdifference between ¹³C and ¹²C can be manifested in very slightdifferences in rates for many reactions and processes. This leads tosmall differences between ¹³C/¹²C ratios for samples subjected tochemical reactions or physical processes. For example, physicalprocesses such as evaporation or diffusion discriminate against heavierisotopes and typically lead to slight enrichment of the heavier isotopein the original sample as the lighter isotope evaporates or diffusesaway more rapidly. The ¹³C/¹²C ratio therefore increases slightly asevaporation or diffusion occurs. For chemical reactions, includingenzymatic reactions, the situation is more complex, but there often is aslight discrimination of one isotope over another, which can be detectedby measuring ¹³C/¹²C ratios or δ¹³C values. For example, atmospheric CO₂can be converted into plant matter via two very different mechanisms forphotosynthesis: the Calvin-Benson pathway, which occurs in C₃ plants,and the Hatch-Slack pathway, which occurs in C₄ plants. These twomechanisms are sufficiently different to produce a measureabledifference in δ¹³C from the same CO₂. For C₄ plants, δ¹³C typicallyranges from −9‰ to −17‰ with a mean near −13‰. For C₃ plants, δ¹³Ctypically ranges from −20‰ to −32‰ with a mean near −27‰. Because theseranges are so different and δ¹³C values can be routinely measured within0.02‰, it is relatively easy to distinguish between plant residuesderived from C₃ versus C₄ plants. This has myriad applications inarcheology and other fields where analysis of carbon-containing residuesfrom cooking or skeletal remains can be used to track the evolution,activities and diets of humans and other animals.

More recently, δ¹³C values have been utilized to detect economic fraud,especially the adulteration of foodstuffs by other materials—includingpotentially harmful synthetics derived from petrochemicals. For example,maize (corn) oil is considered to be a premium vegetable oil and thereis a temptation for unscrupulous producers to dilute maize oil withcheaper oils. Fortunately, maize oil is derived from a C₄ plant whilemost of the cheaper alternatives are derived from C₃ plants or animals.The δ¹³C for authentic maize oil is therefore −13.7‰ to −16.4‰ comparedto −25‰ to −32‰ for the alternatives. Any significant dilution of maizeoil by a cheaper alternative can be detected by measuring δ¹³C.Similarly, the addition of cane sugar (a product of C₄ photosynthesis)to fruit juices, wines, spirits, and honey (all products of C₃photosynthesis) can be detected by measuring δ¹³C values. It is evenpossible to detect the adulteration of natural flavors by syntheticanalogs and the use of illegal synthetic hormone supplements via δ¹³Cvalues.

The current invention utilizes the ability to accurately measure δ¹³Cvalues in order to produce new, isotopically unique isoprenic polymersthat can be readily distinguished from polymers derived frompetroleum-based feedstocks. The current invention also utilizes theability to accurately measure δ¹³C values in order to produce new,isotopically unique isoprenic polymers that can be readily distinguishedfrom natural rubber. A salient feature of the current invention is thatit provides new polymers with a broad range of δ¹³C values that can betailored and subsequently verified for authenticity. As describedearlier, these new polymers satisfy an increasing need from customersfor verifiable products that contain neither potential proteinaceousallergens nor feedstocks derived from petroleum.

The polymers represented by the current invention contain isoprene unitsthat are isotopically unique compared to both natural rubber andsynthetic polymers containing petroleum-derived isoprene. In the case ofnatural rubber derived from Hevea brasiliensis (i.e., the common naturalrubber tree), δ¹³C values typically range from about −27‰ to about −28‰.Guayule rubber, which is derived from a desert shrub, has δ¹³C of about−31‰. Both rubbers exhibit δ¹³C values expected for products of C₃photosynthesis, and both rubbers are known to contain polymer-boundproteins.

Traditional synthetic polyisoprene can have different δ¹³C valuesdepending on the source of isoprene. For isoprene derived fromextractive distillation of C₅ streams from petroleum refineries, δ¹³C isabout −22‰ to about −24‰. This range is typical for light, unsaturatedhydrocarbons derived from petroleum, and polymers containingpetroleum-based isoprene typically contain isoprenic units with the sameδ¹³C. For polymers containing isoprene derived from the reaction ofisobutylene with formaldehyde, δ¹³C values can be about −34.4‰ becauseformaldehyde is often derived from feedstocks with much more negativeδ¹³C values.

The current invention provides isoprene-containing polymers with verydifferent δ¹³C values. For example, fermentation of corn-derived glucose(δ¹³C −10.73‰ with minimal amounts of other carbon-containing nutrients(e.g., yeast extract) produces isoprene which can be polymerized intopolyisoprene with δ¹³C −14.66‰ to −14.85‰. The δ¹³C for this polymerclearly is in a new range that is well outside the normal ranges fornatural rubber and all previously known synthetic polyisoprene, and itis within the range normally associated with products derived from C₄plants. The unique δ¹³C value for this polymer is a direct consequenceof the fact that the isoprene in the polymer is derived from corn-basedglucose, which indeed is a product derived from C₄ plants.

It is recognized by those with ordinary skill in the art that similarresults can be obtained using other sugars or fermentable derived fromC₄ plants. For example, sucrose from sugar cane (δ¹³C −10.4‰, invertsugar from sugar cane (δ¹³C −15.3‰, glucose from cornstarch (δ¹³C−11.1‰, and glucose from hydrolytic degradation of either corn stover(δ¹³C −11.3‰ or sugar cane bagasse (δ¹³C −13.0‰ should all produceisoprene that can be used to produce isoprene polymers with δ¹³C valuesthat are less negative than either natural rubber or synthetic polymerscontaining petroleum-based isoprene. Those with ordinary skill in theart also will recognize that it should be possible to produce isopreneand isoprene polymers with δ¹³C less negative than about −22‰ fromfermentable feedstocks with δ¹³C approximately greater (i.e., lessnegative) than about −18‰, including mixtures of fermentable feedstockswith an average δ¹³C approximately greater than about −18‰.

In addition to producing isoprene-containing polymers with δ¹³C valuescharacteristic of products derived from C₄ plants, those skilled in theart will recognize that uniquely isotopically labeledisoprene-containing polymers can be made from fermentable non-C₄feedstocks. For example, glucose from hydrolyzed softwood pulp (δ¹³C−23‰) should yield isoprene and polyisoprene with δ¹³C near −27‰, whichis in a unique range between the normal ranges observed for isoprenederived from extractive distillation of C₅ fractions and isoprenederived from the reaction of isobutylene with formaldehyde. Thoseskilled in the art also will recognize that fermentation of other sugarswith δ¹³C ranges of approximately −20‰ to about −28‰ should produceisoprene and isoprenic polymers with δ¹³C ranging from about −24‰ toabout −32‰. These other sugars might include (but are not limited to)glucose from hydrolyzed cellulose (δ¹³C −25±2‰), invert sugar from beetsugar (δ¹³C −26‰ to −27‰), and lactose (δ¹³C −27‰ to −28‰. Fermentationof plant oils (δ¹³C −26‰ to −32‰), including palm oil (δ¹³C −30‰) couldprovide access to isoprene polymers with δ¹³C more negative than −30‰.

Those skilled in the art will recognize that cofermentation of two ormore feedstocks can be used to produce isoprene and thereforeisoprene-containing polymers with intermediate δ¹³C values. For example,a 1:1 mixture of sucrose from sugar cane (δ¹³C −10.4‰) and sucrose frombeet sugar (δ¹³C −26‰ to −27‰) should produce isoprene and thereforeisoprene-containing polymers with approximately the same δ¹³C value aspolymer produced from sucrose derived from a single source with theaverage δ¹³C value (i.e., approx −18.5‰). The same should be true forinvert sugars derived from sugar and beets. In both cases, it should beobvious that the same polymers could be synthesized by mixing and then(co)polymerizing equal amounts of isoprene separately prepared fromsucrose or invert sugar derived from sugar cane and beets. It alsoshould be obvious that cofermentation of sugars with other fermentablefeedstocks—such as yeast extract and plant oils—can be used to produceisoprene and therefore isoprene-containing polymers with intermediateδ¹³C values. For example, cofermentation of glucose (δ¹³C −10.73‰) andyeast extract (δ¹³C −26‰ to −27‰) in a ratio of 181.2:17.6 producesisoprene which can be polymerized to polyisoprene with δ¹³C values of−18‰ to −20‰. In contrast, fermentation of glucose with a minimal amountof yeast extract and subsequent polymerization of the isoprene producespolyisoprene with δ¹³C values of −14‰ to −15‰.

For copolymers of isoprene with other monomers, those skilled in the artrecognize that there is a finite amount of isoprene that is incorporatedinto the polymer background as “blocks” of polyisoprene. The tendency ofisoprene to form blocks of two or more isoprenic units—even in “randomcopolymers”—depends on many factors, including the amount of isoprenerelative to other monomers, the type of catalyst used forpolymerization, and the specific reaction conditions for polymerization.The presence of these blocks along the polymer backbone can usually bedetected by NMR spectroscopy. By using a combination of chemicaldegradation (e.g., ozonolysis) and chromatography, it is possible toisolate fragments of these blocks for chemical analysis, includingmeasurement of δ¹³C values for the blocks derived from isoprene. Thisprovides a way for determining whether copolymers of isoprene with othermonomers contain isoprene derived from renewable/sustainable feedstocks,especially feedstocks derived from C₄ plants.

The polyisoprene polymers of this invention which are made with isoprenemonomer from the cells cultures that utilize bio-renewable carbonsources can be identified as such by virtue of their δ¹³C value andother polymer characteristics. For instance, the following isoprenecontaining polymers are verifiable as containing isoprene monomer thatwas produced utilizing the method of this invention:

(1) Polyisoprene polymer which is comprised of repeat units that arederived from isoprene monomer, wherein the polyisoprene polymer has δ¹³Cvalue of greater than −22‰. Such polyisoprene polymers can have a δ¹³Cvalue which is greater than −21‰, and can also have a δ¹³C value whichis greater than −20‰. In some cases, the polyisoprene polymer will has aδ¹³C value which is within the range of −22% to −10‰, and in other casesit will have a δ¹³C value which is within the range of −21% to −12‰. Instill other cases the polyisoprene polymer will have a δ¹³C value whichis within the range of −20‰ to −14‰. In many cases, the polyisoprenepolymer will be polyisoprene homopolymer rubber.

(2) A polyisoprene polymer which is comprised of repeat units that arederived from isoprene monomer, wherein the polyisoprene polymer has δ¹³Cvalue which is within the range of −30‰ to −28.5‰. Such polyisoprenepolymers can have a δ¹³C value which is within the range of −30‰ to−29‰. In some cases, the polyisoprene polymer will have a δ¹³C valuewhich is within the range of −30‰ to −29‰, and in other cases thepolyisoprene polymer will have a δ¹³C value which is within the range of−30% to −29.5‰. In still other cases the polyisoprene polymer can have aδ¹³C value which is within the range of −29.5‰ to −28.5‰ and in stillfurther cases the polyisoprene polymer can have a δ¹³C value which iswithin the range of −29.0‰ to −28.5‰. In many cases, the polyisoprenepolymer will be polyisoprene homopolymer rubber.

(3) A polyisoprene polymer which is comprised of repeat units that arederived from isoprene monomer, wherein the polyisoprene is free ofprotein, and wherein the polyisoprene polymer has δ¹³C value which iswithin the range of −34‰ to −24‰. In some cases this polyisoprenepolymer has δ¹³C value which is within the range of −34‰ to −25‰. Inother cases the polyisoprene polymer has a δ¹³C value which is withinthe range of −33‰ to −25‰, and in still other cases the polyisoprenepolymer has a δ¹³C value which is within the range of −32‰ to −25‰. Inmany cases, the polyisoprene polymer will be polyisoprene homopolymerrubber.

(4) A polyisoprene polymer which is comprised of repeat units that arederived from isoprene monomer, wherein the polyisoprene polymer has acis-1,4-microstructure content of less than 99.9%, wherein thepolyisoprene polymer has a trans-1,4-microstructure content of less than99.9%, and wherein the polyisoprene polymer has δ¹³C value of which iswithin the range of −34‰ to −24‰. Such polyisoprene can have a δ¹³Cvalue which is within the range of −34‰ to −25‰. In some cases thepolyisoprene polymer will have a δ¹³C value which is within the range of−33‰ to −25‰. In other cases the polyisoprene polymer will have a δ¹³Cvalue which is within the range of −32‰ to −25‰. The polyisoprenepolymer can have a cis-1,4-microstructure content of less than 99.8%. Inother cases the polyisoprene polymer will have a cis-1,4-microstructurecontent of less than 99.7%. In still other cases the polyisoprenepolymer will have a cis-1,4-microstructure content of less than 99.5% oreven less than 99%. In many cases the polyisoprene polymer will have acis-1,4-microstructure content of less than 98.5% or even less than 98%.This polyisoprene polymer can also have a polydispersity of less than2.0 or even less than 1.8. In some cases the polyisoprene polymer has apolydispersity of less than 1.6 or even less than 1.5. In still othercases the polyisoprene polymer can have a polydispersity of less than1.4 or even less than 1.2. In many cases the polyisoprene polymer willhave a polydispersity of less than 1.1.

(5) A polyisoprene polymer which is comprised of repeat units that arederived from isoprene monomer, wherein the polyisoprene polymer has a3,4-microstructure content of greater than 2%, and wherein thepolyisoprene polymer has δ¹³C value of which is within the range of −34‰to −24‰. Such polyisoprene polymers can have a δ¹³C value which iswithin the range of −34‰ to −25‰. In some cases the polyisoprene polymerwill have a δ¹³C value which is within the range of −33‰ to −25‰. Inother cases polyisoprene polymer will have a δ¹³C value which is withinthe range of −32‰ to −25‰. The polyisoprene polymer can have a3,4-microstructure content of greater than 5%. In some cases thepolyisoprene polymer will have a 3,4-microstructure content of greaterthan 10%. In other cases the polyisoprene polymer will have a3,4-microstructure content of greater than 15%. In still other thepolyisoprene polymer will have a 3,4-microstructure content of greaterthan 20%. In many cases the polyisoprene polymer will have a3,4-microstructure content of greater than 25%. This polyisoprenepolymer can have a polydispersity of less than 2.0. In some cases thepolyisoprene polymer will have a polydispersity of less than 1.8. Inother cases the polyisoprene polymer will have a polydispersity of lessthan 1.6. In still other cases the polyisoprene polymer will have apolydispersity of less than 1.5 or even than 1.4. In many cases thepolyisoprene polymer will have a polydispersity of less than 1.2 or evenless than 1.1.

(6) A polyisoprene polymer which is comprised of repeat units that arederived from isoprene monomer, wherein the polyisoprene polymer has a1,2-microstructure content of greater than 2%, and wherein thepolyisoprene polymer has δ¹³C value of which is within the range of −34‰to −24‰. Polyisoprene polymers of this type can have a δ¹³C value whichis within the range of −34‰ to −25‰. In some cases, the polyisoprenepolymer will have a δ¹³C value which is within the range of −33‰ to−25‰. In other cases, the polyisoprene polymer will have a δ¹³C valuewhich is within the range of −32‰ to −25‰. The polyisoprene polymer canhave a 1,2-microstructure content of greater then than 5%. In somecases, the polyisoprene polymer will have a 1,2-microstructure contentof greaterthan 10%. In other cases, the polyisoprene polymer will have a1,2-microstructure content of greater than 15%. In still other cases,the polyisoprene polymer will have a 1,2-microstructure content ofgreater than 20%. In many cases, the polyisoprene polymer will have a1,2-microstructure content of greater than 25%. The polyisoprene polymercan have a polydispersity of less than 2.0. In some cases, thepolyisoprene polymer will have a polydispersity of less than 1.8. Inother cases, the polyisoprene polymer will have a polydispersity of lessthan 1.6. In still other cases, the polyisoprene polymer will have apolydispersity of less than 1.5. In many cases, the polyisoprene polymerwill have a polydispersity of less than 1.4 or even less than 1.2. It ispossible for the polyisoprene polymer to have a polydispersity of lessthan 1.1.

(7) A polymer which is comprised of repeat units that are derived fromisoprene monomer and at least one additional monomer, wherein thepolymer includes blocks of repeat units that are derived from isoprene,and wherein the blocks of repeat units that are derived from isoprenehave a δ¹³C value of greater than −22‰. Such polyisoprene polymers canhave a δ¹³C value which is greater than −21‰. In some cases, thepolyisoprene polymer will have a δ¹³C value which is greater than −20‰.In other cases, the polyisoprene polymer will have a δ¹³C value which iswithin the range of −22‰ to −10‰. In still other cases, the polyisoprenepolymer will have a δ¹³C value which is within the range of −21‰ to−12‰. In many cases, the polyisoprene polymer will have a δ¹³C valuethat is within the range of −20‰ to −14‰.

(8) A polymer which is comprised of repeat units that are derived fromisoprene monomer and at least one additional monomer, wherein thepolymer includes blocks of repeat units that are derived from isoprene,and wherein the blocks of repeat units that are derived from isoprenehave a δ¹³C value which is within the range of −34‰ to −24‰. Suchcopolymers can have a δ¹³C value is within the range of −34‰ to −25‰. Insome cases, copolymer of this type will have a δ¹³C value which iswithin the range of −33‰ to −25‰. In other cases, copolymers of thistype will have a δ¹³C value is within the range of −32% to −25‰.Copolymers of this type can be rubbery copolymers of isoprene and1,3-butadiene, rubbery copolymer of isoprene and styrene, rubberycopolymers of isoprene and α-methyl styrene, and the like.

(9) A liquid polyisoprene polymer which is comprised of repeat unitsthat are derived from isoprene monomer, wherein the polyisoprene polymerhas weight average molecular weight which is within the range of 5,000to 100,000, and wherein the liquid polyisoprene polymer has δ¹³C valueof which is within the range of −34‰ to −24‰. Such liquid polyisoprenepolymers can have a δ¹³C value which is within the range of −34% to−25‰. In some cases, the liquid polyisoprene polymer will have a δ¹³Cvalue which is within the range of −33‰ to −25‰. In other cases, theliquid polyisoprene polymer will have a δ¹³C value which is within therange of −32‰ to −25‰. Such liquid polyisoprene polymers can have aweight average molecular weight that is within the range of 20,000 to80,000. In some cases, the liquid polyisoprene polymer will have aweight average molecular weight which is within the range of 30,000 to50,000. In other cases, the polyisoprene polymer will have apolydispersity of less than 2.0 or even less than 1.8. In still othercases, the liquid polyisoprene polymer will have a polydispersity ofless than 1.6 or even less than 1.5. In many cases, the liquidpolyisoprene polymer will have a polydispersity of less than 1.4 or evenless than 1.2. It is possible for the liquid polyisoprene polymer tohave a polydispersity of less than 1.1.

(10) A liquid polyisoprene polymer which is comprised of repeat unitsthat are derived from isoprene monomer, wherein the liquid polyisoprenepolymer has a weight average molecular weight which is within the rangeof 5,000 to 100,000, and wherein the liquid polyisoprene polymer hasδ¹³C value of which is within the range of −34‰ to −24‰. Such liquidpolyisoprene polymers can have a δ¹³C value which is within the range of−34‰ to −25‰. In some cases, the liquid polyisoprene polymer will have aδ¹³C value which is within the range of −33‰ to −25‰. In still othercases, the liquid polyisoprene polymer will have a δ¹³C value which iswithin the range of −32‰ to −25‰. Such liquid polyisoprene can have aweight average molecular weight that is within the range of 20,000 to80,000. The liquid polyisoprene will typically have a weight averagemolecular weight which is within the range of 30,000 to 50,000. Suchliquid polyisoprene can have a polydispersity of less than 2.0. In somecases, the liquid polyisoprene polymer will have a polydispersity ofless than 1.8. In other cases, the liquid polyisoprene polymer has apolydispersity of less than 1.6. In still other cases, the liquidpolyisoprene polymer will have a polydispersity of less than 1.5 or evenless than 1.4. In many cases, the liquid polyisoprene polymer will havea polydispersity of less than 1.2 or even less than 1.1.

This invention is illustrated by the following examples that are merelyfor the purpose of illustration and are not to be regarded as limitingthe scope of the invention or the manner in which it can be practiced.Unless specifically indicated otherwise, parts and percentages are givenby weight.

EXAMPLES

The examples, which are intended to be purely exemplary of the inventionand should therefore not be considered to limit the invention in anyway, also describe and detail aspects and embodiments of the inventiondiscussed above. Unless indicated otherwise, temperature is in degreesCentigrade and pressure is at or near atmospheric pressure. Theforegoing examples and detailed description are offered by way ofillustration and not by way of limitation. All publications, patentapplications, and patents cited in this specification are hereinincorporated by reference as if each individual publication, patentapplication, or patent were specifically and individually indicated tobe incorporated by reference. In particular, all publications citedherein are expressly incorporated herein by reference for the purpose ofdescribing and disclosing compositions and methodologies which might beused in connection with the invention. Although the foregoing inventionhas been described in some detail by way of illustration and example forpurposes of clarity of understanding, it will be readily apparent tothose of ordinary skill in the art in light of the teachings of thisinvention that certain changes and modifications may be made theretowithout departing from the spirit or scope of the appended claims.

In the practice of this invention ¹³C analysis can be done by loading0.5 to 1.0 mg samples into tin cups for carbon isotopic analysis using aCostech ECS4010 Elemental Analyzer as an inlet for a ThermoFinniganDelta Plus XP isotope ratio mass spectrometer. Samples are dropped intoa cobaltous/cobaltic oxide combustion reactor at 1020° C. withcombustion gases being passed in a helium stream at 85mL/min through acopper reactor (650° C.) to convert NO_(x) N₂. CO₂ and N₂ are separatedusing a 3-m 5 Å molecular sieve column. Then, ¹³C/¹²C ratios arecalibrated to the VPDB scale using two laboratory standards (AcetanilideB, −29.52±0.02‰m and cornstarch A, −11.01±0.02‰) which have beencarefully calibrated to the VPDB scale by off-line combustion anddual-inlet analysis using the 2-standard approach of T. B. Coplen et al,New Guidelines for δ¹³C Measurements, Anal. Chem., 78, 2439-2441 (2006).The teachings of Coplen are incorporated herein by reference for thepurpose of teaching the technique for determining δ¹³C values.

Example 1 Production of Isoprene in E. coli Expressing Recombinant KudzuIsoprene Synthase

I. Construction of Vectors for Expression of the Kudzu Isoprene Synthasein E. coli

The protein sequence for the kudzu (Pueraria montana) isoprene synthasegene (IspS) was obtained from GenBank (AAQ84170). A kudzu isoprenesynthase gene, optimized for E. coli codon usage, was purchased fromDNA2.0 (SEQ ID NO:1). The isoprene synthase gene was removed from thesupplied plasmid by restriction endonuclease digestion withBspLU11I/PstI, gel-purified, and ligated into pTrcHis2B (Invitrogen)that had been digested with NcoI/PstI. The construct was designed suchthat the stop codon in the isoprene synthase gene 5′ to the PstI site.As a result, when the construct was expressed the His-Tag is notattached to the isoprene synthase protein. The resulting plasmid,pTrcKudzu, was verified by sequencing (FIGS. 2 and 3).

The isoprene synthase gene was also cloned into pET16b (Novagen). Inthis case, the isoprene synthase gene was inserted into pET16b such thatthe recombinant isoprene synthase protein contained the N-terminal Histag. The isoprene synthase gene was amplified from pTrcKudzu by PCRusing the primer set pET-His-Kudzu-2F:5′-CGTGAGATCATATGTGTGCGACCTCTTCTCAATTTAC (SEQ ID NO:3) andpET-His-Kudzu-R: 5′-CGGTCGACGGATCCCTGCAGTTAGACATACATCAGCTG (SEQ IDNO:4). These primers added an NdeI site at the 5′-end and a BamH1 siteat the 3′ end of the gene respectively. The plasmid pTrcKudzu, describedabove, was used as template DNA, Herculase polymerase (Stratagene) wasused according to manufacture's directions, and primers were added at aconcentration of 10 pMols. The PCR was carried out in a total volume of25 μl. The PCR product was digested with NdeI/BamH1 and cloned intopET16b digested with the same enzymes. The ligation mix was transformedinto E. coli Top10 (Invitrogen) and the correct clone selected bysequencing. The resulting plasmid, in which the kudzu isoprene synthasegene was expressed from the T7 promoter, was designated pETNHisKudzu(FIGS. 4 and 5).

The kudzu isoprene synthase gene was also cloned into the low copynumber plasmid pCL1920. Primers were used to amplify the kudzu isoprenesynthase gene from pTrcKudzu described above. The forward primer added aHindIII site and an E. coli consensus RBS to the 5′ end. The PstIcloning site was already present in pTrcKudzu just 3′ of the stop codonso the reverse primer was constructed such that the final PCR productincludes the PstI site. The sequences of the primers were:HindIII-rbs-Kudzu F: 5′-CATATGAAAGCTTGTATCGATTAAATAAGGAGGAATAAACC (SEQID NO:6) and BamH1-Kudzu R: 5′-CGGTCGACGGATCCCTGCAGTTAGACATACATCAGCTG(SEQ ID NO:4). The PCR product was amplified using Herculase polymerasewith primers at a concentration of 10 pmol and with 1 ng of template DNA(pTrcKudzu). The amplification protocol included 30 cycles of (95° C.for 1 minute, 60° C. for 1 minute, 72° C. for 2 minutes). The productwas digested with HindIII and PstI and ligated into pCL1920 which hadalso been digested with HindIII and PstI. The ligation mix wastransformed into E. coli Top10. Several transformants were checked bysequencing. The resulting plasmid was designated pCL-lac-Kudzu (FIGS. 6and 7).

II. Determination of Isoprene Production

For the shake flask cultures, one ml of a culture was transferred fromshake flasks to 20 ml CTC headspace vials (Agilent vial cat# 5188 2753;cap cat# 5188 2759). The cap was screwed on tightly and the vialsincubated at the equivalent temperature with shaking at 250 rpm. After30 minutes the vials were removed from the incubator and analyzed asdescribed below (see Table 1 for some experimental values from thisassay).

In cases where isoprene production in fermentors was determined, sampleswere taken from the off-gas of the fermentor and analyzed directly asdescribed below (see Table 2 for some experimental values from thisassay).

The analysis was performed using an Agilent 6890 GC/MS system interfacedwith a CTC Analytics (Switzerland) CombiPAL autosampler operating inheadspace mode. An Agilent HP-5MS GC/MS column (30 m×0.25 mm; 0.25 μmfilm thickness) was used for separation of analytes. The sampler was setup to inject 500 μL of headspace gas. The GC/MS method utilized heliumas the carrier gas at a flow of 1 ml/minutes. The injection port washeld at 250° C. with a split ratio of 50:1. The oven temperature washeld at 37° C. for the 2 minute duration of the analysis. The Agilent5793N mass selective detector was run in single ion monitoring (SIM)mode on m/z 67. The detector was switched off from 1.4 to 1.7 minutes toallow the elution of permanent gases. Under these conditions isoprene(2-methyl-1,3-butadiene) was observed to elute at 1.78 minutes. Acalibration table was used to quantify the absolute amount of isopreneand was found to be linear from 1 μg/L to 200 μg/L. The limit ofdetection was estimated to be 50 to 100 ng/L using this method.

III. Production of Isoprene in Shake Flasks Containing E. coli CellsExpressing Recombinant Isoprene Synthase

The vectors described above were introduced to E. coli strain BL21(Novagen) to produce strains BL21/ptrcKudzu, BL21/pCL-lac-Kudzu andBL21/pETHisKudzu. The strains were spread for isolation onto LA (Luriaagar) and carbenicillin (50 μg/ml) and incubated overnight at 37° C.Single colonies were inoculated into 250 ml baffled shake flaskscontaining 20 ml Luria Bertani broth (LB) and carbenicillin (100 μg/ml).Cultures were grown overnight at 20° C. with shaking at 200 rpm. TheOD₆₀₀ of the overnight cultures were measured and the cultures werediluted into a 250 ml baffled shake flask containing 30 ml MagicMedia(Invitrogen) and carbenicillin (100 μg/ml) to an OD₆₀₀˜0.05. The culturewas incubated at 30° C. with shaking at 200 rpm. When the OD₆₀₀˜0.5-0.8,400 μM IPTG was added and the cells were incubated for a further 6 hoursat 30° C. with shaking at 200 rpm. At 0, 2, 4 and 6 hours afterinduction with IPTG, 1 ml aliquots of the cultures were collected, theOD₆₀₀ was determined and the amount of isoprene produced was measured asdescribed above. Results are shown in FIG. 8.

IV. Production of Isoprene from BL21/ptrcKudzu in 14 Liter Fermentation

Large scale production of isoprene from E. coli containing therecombinant kudzu isoprene synthase gene was determined from a fed-batchculture. The recipe for the fermentation media (TM2) per liter offermentation medium was as follows: K₂HPO₄ 13.6 g, KH₂PO₄ 13.6 g,MgSO₄*7H₂O 2 g, citric acid monohydrate 2 g, ferric ammonium citrate 0.3g, (NH₄)₂SO₄ 3.2 g, yeast extract 5 g, 1000× Modified Trace MetalSolution 1 ml. All of the components were added together and dissolvedin diH₂O. The pH was adjusted to 6.8 with potassium hydroxide (KOH) andq.s. to volume. The final product was filter sterilized with 0.22μfilter (only, not autoclaved). The recipe for 1000× Modified Trace MetalSolution was as follows: Citric Acids*H₂O 40 g, MnSO₄*H₂O 30 g, NaCl 10g, FeSO₄*7H₂O 1 g, CoCl₂*6H₂O 1 g, ZnSO*7H₂O 1 g, CuSO₄*5H₂O 100 mg,H₃BO₃ 100 mg, NaMoO₄*2H₂O 100 mg. Each component was dissolved one at atime in diH₂O, pH to 3.0 with HCl/NaOH, then q.s. to volume and filtersterilized with a 0.22μ filter.

This experiment was carried out in 14 L bioreactor to monitor isopreneformation from glucose at the desired fermentation, pH 6.7 andtemperature 34° C. An inoculum of E. coli strain BL21/ptrcKudzu takenfrom a frozen vial was prepared in soytone-yeast extract-glucose medium.After the inoculum grew to OD₅₅₀=0.6, two 600 ml flasks were centrifugedand the contents resuspended in 70 ml supernatant to transfer the cellpellet (70 ml of OD 3.1 material) to the bioreactor. At various timesafter inoculation, samples were removed and the amount of isopreneproduced was determined as described above. Results are shown in FIG. 9.

Example 2 Production of Isoprene in E. coli Expressing RecombinantPoplar Isoprene Synthase

The protein sequence for the poplar (Populus alba×Populus tremula)isoprene synthase (Schnitzler, J-P, et al. (2005) Planta 222:777-786)was obtained from GenBank (CAC35696). A gene, codon optimized for E.coli, was purchased from DNA2.0 (p9796-poplar, FIGS. 30 and 31). Theisoprene synthase gene was removed from the supplied plasmid byrestriction endonuclease digestion with BspLU11I/PstI, gel-purified, andligated into pTrcHis2B that had been digested with NcoI/PstI. Theconstruct is cloned such that the stop codon in the insert is before thePstI site, which results in a construct in which the His-Tag is notattached to the isoprene synthase protein. The resulting plasmidpTrcPoplar (FIGS. 32 and 33), was verified by sequencing.

Example 3 Production of Isoprene in Panteoa citrea ExpressingRecombinant Kudzu Isoprene Synthase

The pTrcKudzu and pCL-lac Kudzu plasmids described in Example 1 wereelectroporated into P. citrea (U.S. Pat. No. 7,241,587). Transformantswere selected on LA containing carbenicillin (200 μg/ml) orspectinomycin (50 μg/ml) respectively. Production of isoprene from shakeflasks and determination of the amount of isoprene produced wasperformed as described in Example 1 for E. coli strains expressingrecombinant kudzu isoprene synthase. Results are shown in FIG. 10.

Example 4 Production of Isoprene in Bacillus subtilis ExpressingRecombinant Kudzu Isoprene Synthase

I. Construction of a B. subtilis Replicating Plasmid for the Expressionof Kudzu Isoprene Synthase

The kudzu isoprene synthase gene was expressed in Bacillus subtilisaprEnprE Pxyl-comK strain (BG3594comK) using a replicating plasmid(pBS19 with a chloramphenicol resistance cassette) under control of theaprE promoter. The isoprene synthase gene, the aprE promoter and thetranscription terminator were amplified separately and fused using PCR.The construct was then cloned into pBS19 and transformed into B.subtilis.

a) Amplification of the aprE Promoter

The aprE promoter was amplified from chromosomal DNA from Bacillussubtilis using the following primers:

CF 797 (+) Start aprE promoter MfeI (SEQ ID NO: 58)5′- GACATCAATTGCTCCATTTTCTTCTGCTATCCF 07-43 (−) Fuse aprE promoter to Kudzu ispS (SEQ ID NO: 59)5′- ATTGAGAAGAGGTCGCACACACTCTTTACCCTCTCCTTTTAb) Amplification of the Isoprene Synthase Gene

The kudzu isoprene synthase gene was amplified from plasmid pTrcKudzu(SEQ ID NO:2). The gene had been codon optimized for E. coli andsynthesized by DNA 2.0. The following primers were used:

CF 07-42 (+) Fuse the aprE promoter to kudzuisoprene synthase gene (GTG start codon) (SEQ ID NO: 60)5′-TAAAAGGAGAGGGTAAAGAGTGTGTGCGACCTCTTCTCAAT CF 07-45 (−) Fuse the 3′end of kudzu isoprene synthase gene to the terminator (SEQ ID NO: 61)5′-CCAAGGCCGGTTTTTTTTAGACATACATCAGCTGGTTAATCc) Amplification of the Transcription Terminator

The terminator from the alkaline serine protease of Bacillusamyliquefaciens was amplified from a previously sequenced plasmidpJHPms382 using the following primers:

CF 07-44 (+) Fuse the 3′ end of kudzu isoprenesynthase to the terminator (SEQ ID NO: 62)5′-GATTAACCAGCTGATGTATGTCTAAAAAAAACCGGCCTTGGCF 07-46 (−) End of B. amyliquefaciens terminator (BamHI)(SEQ ID NO: 63) 5′-GACATGACGGATCCGATTACGAATGCCGTCTC

The kudzu fragment was fused to the terminator fragment using PCR withthe following primers:

CF 07-42 (+) Fuse the aprE promoter to kudzuisoprene synthase gene (GTG start codon) (SEQ ID NO: 60)5′-TAAAAGGAGAGGGTAAAGAGTGTGTGCGACCTCTTCTCAATCF 07-46 (−) End of B. amyliquefaciens terminator (BamHI)(SEQ ID NO: 63) 5′-GACATGACGGATCCGATTACGAATGCCGTCTC

The kudzu-terminator fragment was fused to the promoter fragment usingPCR with the following primers:

CF 797 (+) Start aprE promoter MfeI (SEQ ID NO: 64)5′-GACATCAATTGCTCCATTTTCTTCTGCTATCCF 07-46 (−) End of B. amyliquefaciens terminator (BamHI)(SEQ ID NO: 63) 5′-GACATGACGGATCCGATTACGAATGCCGTCTC

The fusion PCR fragment was purified using a Qiagen kit and digestedwith the restriction enzymes MfeI and BamHI. This digested DNA fragmentwas gel purified using a Qiagen kit and ligated to a vector known aspBS19, which had been digested with EcoRI and BamHI and gel purified.

The ligation mix was transformed into E. coli Top 10 cells and colonieswere selected on LA and 50 carbenicillin plates. A total of six colonieswere chosen and grown overnight in LB and 50 carbenicillin and thenplasmids were isolated using a Qiagen kit. The plasmids were digestedwith EcoRI and BamHI to check for inserts and three of the correctplasmids were sent in for sequencing with the following primers:

CF 149 (+) EcoRI start of aprE promoter (SEQ ID NO: 65)5′-GACATGAATTCCTCCATTTTCTTCTGCCF 847 (+) Sequence in pXX 049 (end of aprE promoter) (SEQ ID NO: 66)5′-AGGAGAGGGTAAAGAGTGAG CF 07-45 (−) Fuse the 3′ end of kudzu isoprenesynthase to the terminator (SEQ ID NO: 61)5′-CCAAGGCCGGTTTTTTTTAGACATACATCAGCTGGTTAATCCF 07-48 (+) Sequencing primer for kudzu isoprene synthase(SEQ ID NO: 67) 5′-CTTTTCCATCACCCACCTGAAGCF 07-49 (+) Sequencing in kudzu isoprene synthase (SEQ ID NO: 68)5′-GGCGAAATGGTCCAACAACAAAATTATC

The plasmid designated pBS Kudzu #2 (FIGS. 52 and 12) was correct bysequencing and was transformed into BG 3594 comK, a Bacillus subtilishost strain. Selection was done on LA and 5 chloramphenicol plates. Atransformant was chosen and struck to single colonies on LA and 5chloramphenicol, then grown in LB and 5 chloramphenicol until it reachedan OD₆₀₀ of 1.5. It was stored frozen in a vial at −80° C. in thepresence of glycerol. The resulting strain was designated CF 443.

II. Production of Isoprene in Shake Flasks Containing B. subtilis CellsExpressing Recombinant Isoprene Synthase.

Overnight cultures were inoculated with a single colony of CF 443 from aLA and Chloramphenicol (Cm, 25 μg/ml). Cultures were grown in LB and Cmat 37° C. with shaking at 200 rpm. These overnight cultures (1 ml) wereused to inoculate 250 ml baffled shake flasks containing 25 ml Grants IImedia and chloramphenicol at a final concentration of 25 μg/ml. GrantsII Media recipe was 10 g soytone, 3 ml 1M K₂HPO₄, 75 g glucose, 3.6 gurea, 100 ml 10×MOPS, q.s. to 1 L with H₂O, pH 7.2; 10×MOPS recipe was83.72 g MOPS, 7.17 g tricine, 12 g KOH pellets, 10 ml 0.276M K₂SO₄solution, 10 ml 0.528M MgCl₂ solution, 29.22 g NaCl, 100 ml 100×micronutrients, q.s. to 1 L with H₂O; and 100× micronutrients recipe was1.47 g CaCl₂*2H₂O, 0.4 g FeSO₄.7H₂O, 0.1 g MnSO₄*H₂O, 0.1 g ZnSO₄*H₂O,0.05 g CuCl₂*2H₂O, 0.1 g CoCl₂*6H₂O, 0.1 g Na₂MoO₄.2H₂O, q.s. to 1 Lwith H₂O, Shake flasks were incubated at 37° C. and samples were takenat 18, 24, and 44 hours. At 18 hours the headspaces of CF443 and thecontrol strain were sampled. This represented 18 hours of accumulationof isoprene. The amount of isoprene was determined by gas chromatographyas described in Example 1. Production of isoprene was enhancedsignificantly by expressing recombinant isoprene synthase (FIG. 11).

III. Production of Isoprene by CF443 in 14 L Fermentation

Large scale production of isoprene from B. subtilis containing therecombinant kudzu isoprene synthase gene on a replication plasmid wasdetermined from a fed-batch culture. Bacillus strain CF 443, expressinga kudzu isoprene synthase gene, or control stain which does not expressa kudzu isoprene synthase gene were cultivated by conventional fed-batchfermentation in a nutrient medium containing soy meal (Cargill), sodiumand potassium phosphate, magnesium sulfate and a solution of citricacid, ferric chloride and manganese chloride. Prior to fermentation themedia is macerated for 90 minutes using a mixture of enzymes includingcellulases, hemicellulases and pectinases (see, WO95/04134). 14-L batchfermentations are fed with 60% wt/wt glucose (Cargill DE99 dextrose, ADMVersadex greens or Danisco invert sugar) and 99% wt/wt oil (WesternFamily soy oil, where the 99% wt/wt is the concentration of oil beforeit was added to the cell culture medium). Feed was started when glucosein the batch was non-detectable. The feed rate was ramped over severalhours and was adjusted to add oil on an equal carbon basis. The pH wascontrolled at 6.8-7.4 using 28% w/v ammonium hydroxide. In case offoaming, antifoam agent was added to the media. The fermentationtemperature was controlled at 37° C. and the fermentation culture wasagitated at 750 rpm. Various other parameters such as pH, DO %, airflow,and pressure were monitored throughout the entire process. The DO % ismaintained above 20. Samples were taken over the time course of 36 hoursand analyzed for cell growth (OD₅₅₀) and isoprene production. Results ofthese experiments are presented in FIGS. 53A and 53B.

IV. Integration of the Kudzu Isoprene Synthase (ispS) in B. subtilis.

The kudzu isoprene synthase gene was cloned in an integrating plasmid(pJH101-cmpR) under the control of the aprE promoter. Under theconditions tested, no isoprene was detected.

Example 5 Production of Isoprene in Trichoderma

I. Construction of Vectors for Expression of the Kudzu Isoprene Synthasein Trichoderma reesei

The Yarrowia lipolytica codon-optimized kudzu IS gene was synthesized byDNA 2.0 (SEQ ID NO:8) (FIG. 13). This plasmid served as the template forthe following PCR amplification reaction: 1 μl plasmid template (20ng/ul), 1 μl Primer EL-945 (10 uM)5′-GCTTATGGATCCTCTAGACTATTACACGTACATCAATTGG (SEQ ID NO:9), 1 μl PrimerEL-965 (10 uM) 5′-CACCATGTGTGCAACCTCCTCCCAGTTTAC (SEQ ID NO:10), 1 μldNTP (10 mM), 5 μl 10× PfuUltra II Fusion HS DNA Polymerase Buffer, 1 μlPfuUltra II Fusion HS DNA Polymerase, 40 μl water in a total reactionvolume of 50 μl. The forward primer contained an additional 4nucleotides at the 5′-end that did not correspond to the Y. lipolyticacodon-optimized kudzu isoprene synthase gene, but was required forcloning into the pENTR/D-TOPO vector. The reverse primer contained anadditional 21 nucleotides at the 5′-end that did not correspond to theY. lipolytica codon-optimized kudzu isoprene synthase gene, but wereinserted for cloning into other vector backbones. Using the MJ ResearchPTC-200 Thermocycler, the PCR reaction was performed as follows: 95° C.for 2 minutes (first cycle only), 95° C. for 30 seconds, 55° C. for 30seconds, 72° C. for 30 seconds (repeat for 27 cycles), 72° C. for 1minute after the last cycle. The PCR product was analyzed on a 1.2%E-gel to confirm successful amplification of the Y. lipolyticacodon-optimized kudzu isoprene synthase gene.

The PCR product was then cloned using the TOPO pENTR/D-TOPO Cloning Kitfollowing manufacturer's protocol: 1 μl PCR reaction, 1 μl Saltsolution, 1 μl TOPO pENTR/D-TOPO vector and 3 μl water in a totalreaction volume of 6 μl. The reaction was incubated at room temperaturefor 5 minutes. One microliter of TOPO reaction was transformed intoTOP10 chemically competent E. coli cells. The transformants wereselected on LA and 50 μg/ml kanamycin plates. Several colonies werepicked and each was inoculated into a 5 ml tube containing LB and 50μg/ml kanamycin and the cultures grown overnight at 37° C. with shakingat 200 rpm. Plasmids were isolated from the overnight culture tubesusing QIAprep Spin Miniprep Kit, following manufacturer's protocol.Several plasmids were sequenced to verify that the DNA sequence wascorrect.

A single pENTR/D-TOPO plasmid, encoding a Y. lipolytica codon-optimizedkudzu isoprene synthase gene, was used for Gateway Cloning into acustom-made pTrex3g vector. Construction of pTrex3g is described in WO2005/001036 A2. The reaction was performed following manufacturer'sprotocol for the Gateway LR Clonase II Enzyme Mix Kit (Invitrogen): 1 μlY. lipolytica codon-optimized kudzu isoprene synthase gene pENTR/D-TOPOdonor vector, 1 μl pTrex3g destination vector, 6 μl TE buffer, pH 8.0 ina total reaction volume of 8 μl. The reaction was incubated at roomtemperature for 1 hour and then 1 μl proteinase K solution was added andthe incubation continued at 37° C. for 10 minutes. Then 1 μl of reactionwas transformed into TOP10 chemically competent E. coli cells. Thetransformants were selected on LA and 50 μg/ml carbenicillin plates.Several colonies were picked and each was inoculated into a 5 ml tubecontaining LB and 50 μg/ml carbenicillin and the cultures were grownovernight at 37° C. with shaking at 200 rpm. Plasmids were isolated fromthe overnight culture tubes using QIAprep Spin Miniprep Kit (Qiagen,Inc.), following manufacturer's protocol. Several plasmids weresequenced to verify that the DNA sequence was correct.

Biolistic transformation of Y lipolytica codon-optimized kudzu isoprenesynthase pTrex3g plasmid (FIG. 14) into a quad delete Trichoderma reeseistrain was performed using the Biolistic PDS-1000/HE Particle DeliverySystem (see WO 2005/001036 A2).

Isolation of stable transformants and shake flask evaluation wasperformed using protocol listed in Example 11 of patent publication WO2005/001036 A2.

II. Production of Isoprene in Recombinant Strains of T. reesei

One ml of 15 and 36 hour old cultures of isoprene synthase transformantsdescribed above were transferred to head space vials. The vials weresealed and incubated for 5 hours at 30° C. Head space gas was measuredand isoprene was identified by the method described in Example 1. Two ofthe transformants showed traces of isoprene. The amount of isoprenecould be increased by a 14 hour incubation. The two positive samplesshowed isoprene at levels of about 0.5 μg/L for the 14 hour incubation.The untransformed control showed no detectable levels of isoprene. Thisexperiment shows that T. reesei is capable of producing isoprene fromendogenous precursor when supplied with an exogenous isoprene synthase.

Example 6 Production of Isoprene in Yarrowia

I. Construction of Vectors for Expression of the Kudzu Isoprene Synthasein Yarrowia lipolytica.

The starting point for the construction of vectors for the expression ofthe kudzu isoprene synthase gene in Yarrowia lipolytica was the vectorpSPZ1(MAP29Spb). The complete sequence of this vector (SEQ ID No:11) isshown in FIG. 15.

The following fragments were amplified by PCR using chromosomal DNA of aY lipolytica strain GICC 120285 as the template: a promotorless form ofthe URA3 gene, a fragment of 18S ribosomal RNA gene, a transcriptionterminator of the Y. lipolytica XPR2 gene and two DNA fragmentscontaining the promoters of XPR2 and ICL1 genes. The following PCRprimers were used:

ICL1 3 (SEQ ID NO: 69) 5′-GGTGAATTCAGTCTACTGGGGATTCCCAAATCTATATATACTGCAGGTGAC ICL1 5 (SEQ ID NO: 70) 5′-GCAGGTGGGAAACTATGCACTCC XPR 3(SEQ ID NO: 71) 5′-CCTGAATTCTGTTGGATTGGAGGATTGGATAGTGGG XPR 5(SEQ ID NO: 72) 5′-GGTGTCGACGTACGGTCGAGCTTATTGACC XPRT3 (SEQ ID NO: 73)5′-GGTGGGCCCGCATTTTGCCACCTACAAGCCAG XPRT 5 (SEQ ID NO: 74)5′-GGTGAATTCTAGAGGATCCCAACGCTGTTGCCTACAACGG Y18S3 (SEQ ID NO: 75)5′-GGTGCGGCCGCTGTCTGGACCTGGTGAGTTTCCCCG Y18S 5 (SEQ ID NO: 76)5′-GGTGGGCCCATTAAATCAGTTATCGTTTATTTGATAG YURA3 (SEQ ID NO: 77)5′-GGTGACCAGCAAGTCCATGGGTGGTTTGATCATGG YURA 50 (SEQ ID NO: 78)5′-GGTGCGGCCGCCTTTGGAGTACGACTCCAACTATG YURA 51 (SEQ ID NO: 79)5′-GCGGCCGCAGACTAAATTTATTTCAGTCTCC

For PCR amplification the PfuUltraII polymerase (Stratagene),supplier-provided buffer and dNTPs, 2.5 μM primers and the indicatedtemplate DNA were used in accordance with the manufacturer'sinstructions. The amplification was done using the following cycle: 95°C. for 1 min; 34× (95° C. for 30 sec; 55° C. for 30 sec; 72° C. for 3min) and 10 min at 72° C. followed by a 4° C. incubation.

Synthetic DNA molecules encoding the kudzu isoprene synthase gene,codon-optimized for expression in Yarrowia, was obtained from DNA 2.0(FIG. 16; SEQ ID NO:12). Full detail of the construction scheme of theplasmids pYLA(KZ1) and pYLI(KZ1) carrying the synthetic kudzu isoprenesynthase gene under control of XPR2 and ICL1 promoters respectively ispresented in FIG. 18. Control plasmids in which a mating factor gene(MAP29) is inserted in place of an isoprene synthase gene were alsoconstructed (FIGS. 18E and 18F).

A similar cloning procedure can be used to express a poplar (Populusalba×Populus tremula) isoprene synthase gene. The sequence of the poplarisoprene is described in Miller B. et al. (2001) Planta 213, 483-487 andshown in FIG. 17 (SEQ ID NO:13). A construction scheme for thegeneration the plasmids pYLA(POP1) and pYLI(POP1) carrying syntheticpoplar isoprene synthase gene under control of XPR2 and ICL1 promotersrespectively is presented in FIGS. 18A and B.

II. Production of Isoprene by Recombinant Strains of Y. lipolytica.

Vectors pYLA(KZ1), pYLI(KZ1), pYLA(MAP29) and pYLI(MAP29) were digestedwith Sad and used to transform the strain Y. lipolytica CLIB 122 by astandard lithium acetate/polyethylene glycol procedure to uridineprototrophy. Briefly, the yeast cells grown in YEPD (1% yeast extract,2% peptone, 2% glucose) overnight, were collected by centrifugation(4000 rpm, 10 min), washed once with sterile water and suspended in 0.1M lithium acetate, pH 6.0. Two hundred μl aliquots of the cellsuspension were mixed with linearized plasmid DNA solution (10-20 μg),incubated for 10 minutes at room temperature and mixed with 1 ml of 50%PEG 4000 in the same buffer. The suspensions were further incubated for1 hour at room temperature followed by a 2 minutes heat shock at 42° C.Cells were then plated on SC his leu plates (0.67% yeast nitrogen base,2% glucose, 100 mg/L each of leucine and histidine). Transformantsappeared after 3-4 days of incubation at 30° C.

Three isolates from the pYLA(KZ1) transformation, three isolates fromthe pYLI(KZ1) transformation, two isolates from the pYLA(MAP29)transformation and two isolates from the pYLI(MAP29) transformation weregrown for 24 hours in YEP7 medium (1% yeast extract, 2% peptone, pH 7.0)at 30° C. with shaking. Cells from 10 ml of culture were collected bycentrifugation, resuspended in 3 ml of fresh YEP7 and placed into 15 mlscrew cap vials. The vials were incubated overnight at room temperaturewith gentle (60 rpm) shaking. Isoprene content in the headspace of thesevials was analyzed by gas chromatography using mass-spectrometricdetector as described in Example 1. All transformants obtained withpYLA(KZ1) and pYLI(KZ1) produced readily detectable amounts of isoprene(0.5 μg/L to 1 μg/L, FIG. 20). No isoprene was detected in the headspaceof the control strains carrying phytase gene instead of an isoprenesynthase gene.

Example 7 Production of Isoprene in E. coli Expressing Kudzu IsopreneSynthase and idi, or dxs, or idi and dxs

I. Construction of Vectors Encoding Kudzu Isoprene Synthase and idi, ordxs, or idi and dxs for the Production of Isoprene in E. coli

i) Construction of pTrcKudzuKan

The bla gene of pTrcKudzu (described in Example 1) was replaced with thegene conferring kanamycin resistance. To remove the bla gene, pTrcKudzuwas digested with BspHI, treated with Shrimp Alkaline Phosphatase (SAP),heat killed at 65° C., then end-filled with Klenow fragment and dNTPs.The 5 kbp large fragment was purified from an agarose gel and ligated tothe kan^(r) gene which had been PCR amplified from pCR-Blunt-II-TOPOusing primers MCM22 5′-GATCAAGCTTAACCGGAATTGCCAGCTG (SEQ ID NO:14) andMCM23 5′-GATCCGATCGTCAGAAGAACTCGTCAAGAAGGC (SEQ ID NO:15), digested withHindIII and Pvul, and end-filled. A transformant carrying a plasmidconferring kanamycin resistance (pTrcKudzuKan) was selected on LAcontaining kanamycin 50 μg/ml.

ii) Construction of pTrcKudzu yIDI Kan

pTrcKudzuKan was digested with PstI, treated with SAP, heat killed andgel purified. It was ligated to a PCR product encoding idi from S.cerevisiae with a synthetic RBS. The primers for PCR were NsiI-YIDI 1 F5′-CATCAATGCATCGCCCTTAGGAGGTAAAAAAAAATGAC (SEQ ID NO:16) and PstI-YIDI1R 5′-CCTTCTGCAGGACGCGTTGTTATAGC (SEQ ID NO:17); and the template was S.cerevisiae genomic DNA. The PCR product was digested with NsiI and PstIand gel purified prior to ligation. The ligation mixture was transformedinto chemically competent TOP10 cells and selected on LA containing 50μg/ml kanamycin. Several transformants were isolated and sequenced andthe resulting plasmid was called pTrcKudzu-yIDI(kan) (FIGS. 34 and 35).

iii) Construction of pTrcKudzu DXS Kan

Plasmid pTrcKudzuKan was digested with PstI, treated with SAP, heatkilled and gel purified. It was ligated to a PCR product encoding dxsfrom E. coli with a synthetic RBS.

The primers for PCR were MCM135′-GATCATGCATTCGCCCTTAGGAGGTAAAAAAACATGAGTTTTGATATTGCCAAA TACCCG (SEQ IDNO:18) and MCM14 5′-CATGCTGCAGTTATGCCAGCCAGGCCTTGAT (SEQ ID NO:19); andthe template was E. coli genomic DNA. The PCR product was digested withNsiI and PstI and gel purified prior to ligation. The resultingtransformation reaction was transformed into TOP10 cells and selected onLA with kanamycin 50 μg/ml. Several transformants were isolated andsequenced and the resulting plasmid was called pTrcKudzu-DXS(kan) (FIGS.36 and 37).

iv) Construction of pTrcKudzu-yIDI-dxs (kan)

pTrcKudzu-yIDI(kan) was digested with PstI, treated with SAP, heatkilled and gel purified. It was ligated to a PCR product encoding E.coli dxs with a synthetic RBS (primers MCM135′-GATCATGCATTCGCCCTTAGGAGGTAAAAAAACATGAGTTTTGATATTGCCAAA TACCCG (SEQ IDNO:18) and MCM14 5′-CATGCTGCAGTTATGCCAGCCAGGCCTTGAT (SEQ ID NO:19);template TOP10 cells) which had been digested with NsiI and PstI and gelpurified. The final plasmid was called pTrcKudzu-yIDI-dxs (kan) (FIGS.21 and 22).

v) Construction of pCL PtrcKudzu

A fragment of DNA containing the promoter, structural gene andterminator from Example 1 above was digested from pTrcKudzu using Sspland gel purified. It was ligated to pCL1920 which had been digested withPvuII, treated with SAP and heat killed. The resulting ligation mixturewas transformed into TOP10 cells and selected in LA containingspectinomycin 50 μg/ml. Several clones were isolated and sequenced andtwo were selected. pCL PtrcKudzu and pCL PtrcKudzu (A3) have the insertin opposite orientations (FIGS. 38-41).

vi) Construction of pCL PtrcKudzu yIDI

The NsiI-PstI digested, gel purified, IDI PCR amplicon from (ii) abovewas ligated into pCL PtrcKudzu which had been digested with PstI,treated with SAP, and heat killed. The ligation mixture was transformedinto TOP10 cells and selected in LA containing spectinomycin 50 μg/ml.Several clones were isolated and sequenced and the resulting plasmid iscalled pCL PtrcKudzu yIDI (FIGS. 42 and 43).

vii) Construction of pCL PtrcKudzu DXS

The NsiI-PstI digested, gel purified, DXS PCR amplicon from (iii) abovewas ligated into pCL PtrcKudzu (A3) which had been digested with PstI,treated with SAP, and heat killed. The ligation mixture was transformedinto TOP10 cells and selected in LA containing spectinomycin 50 μg/ml.Several clones were isolated and sequenced and the resulting plasmid iscalled pCL PtrcKudzu DXS (FIGS. 44 and 45).

II. Measurement of Isoprene in Headspace from Cultures Expressing KudzuIsoprene Synthase, idi, and/or dxs at Different Copy Numbers.

Cultures of E. coli BL21(λADE3) previously transformed with plasmidspTrcKudzu(kan) (A), pTrcKudzu-yIDI kan (B), pTrcKudzu-DXS kan (C),pTrcKudzu-yIDI-DXS kan (D) were grown in LB kanamycin 50 μg/mL. Culturesof pCL PtrcKudzu (E), pCL PtrcKudzu, pCL PtrcKudzu-yIDI (F) and pCLPtrcKudzu-DXS (G) were grown in LB spectinomycin 50 μg/mL. Cultures wereinduced with 400 μM IPTG at time 0 (OD₆₀₀ approximately 0.5) and samplestaken for isoprene headspace measurement (see Example 1). Results areshown in FIG. 23A-23G.

Plasmid pTrcKudzu-yIDI-dxs (kan) was introduced into E. coli strain BL21by transformation. The resulting strain BL21/pTrc Kudzu IDI DXS wasgrown overnight in LB containing kanamycin (50 μg/ml) at 20° C. and usedto inoculate shake flasks of TM3 (13.6 g K₂PO₄, 13.6 g KH₂PO₄, 2.0 gMgSO₄.7H₂O), 2.0 g citric acid monohydrate, 0.3 g ferric ammoniumcitrate, 3.2 g (NH₄)₂SO₄, 0.2 g yeast extract, 1.0 ml 1000× ModifiedTrace Metal Solution, adjusted to pH 6.8 and q.s. to H₂O, and filtersterilized) containing 1% glucose. Flasks were incubated at 30° C. untilan OD₆₀₀ of 0.8 was reached, and then induced with 400 μM IPTG. Sampleswere taken at various times after induction and the amount of isoprenein the head space was measured as described in Example 1. Results areshown in FIG. 23H.

III. Production of Isoprene from Biomass in E. coli/pTrcKudzu yIDI DXS

The strain BL21 pTrcKudzuIDIDXS was tested for the ability to generateisoprene from three types of biomass; bagasse, corn stover and soft woodpulp with glucose as a control. Hydrolysates of the biomass wereprepared by enzymatic hydrolysis (Brown, L. and Torget, R., 1996, NRELstandard assay method Lap-009 “Enzymatic Saccharification ofLignocellulosic Biomass”) and used at a dilution based upon glucoseequivalents. In this example, glucose equivalents were equal to 1%glucose. A single colony from a plate freshly transformed cells of BL21(DE3) pTrcKudzu yIDI DXS (kan) was used to inoculate 5 ml of LB pluskanamycin (50 μg/ml). The culture was incubated overnight at 25° C. withshaking. The following day the overnight culture was diluted to an OD₆₀₀of 0.05 in 25 ml of TM3 and 0.2% YE and 1% feedstock. The feedstock wascorn stover, bagasse, or softwood pulp. Glucose was used as a positivecontrol and no glucose was used as a negative control. Cultures wereincubated at 30° C. with shaking at 180 rpm. The culture was monitoredfor OD₆₀₀ and when it reached an OD₆₀₀ of ˜0.8, cultures were analyzedat 1 and 3 hours for isoprene production as described in Example 1.Cultures are not induced. All cultures containing added feedstockproduce isoprene equivalent to those of the glucose positive control.Experiments were done in duplicate and are shown in FIG. 46.

IV. Production of Isoprene from Invert Sugar in E. coli/pTrcKudzuIDIDXS

A single colony from a plate freshly transformed cells of BL21(λDE3)/pTrcKudzu yIDI DXS (kan) was used to inoculate 5 mL of LB andkanamycin (50 μg/ml). The culture was incubated overnight at 25° C. withshaking. The following day the overnight culture was diluted to an OD₆₀₀of 0.05 in 25 ml of TM3 and 0.2% YE and 1% feedstock. Feedstock wasglucose, inverted glucose or corn stover. The invert sugar feedstock(Danisco Invert Sugar) was prepared by enzymatically treating sucrosesyrup. AFEX corn stover was prepared as described below (Part V). Thecells were grown at 30° C. and the first sample was measured when thecultures reached an OD₆₀₀˜0.8-1.0 (0 hour). The cultures were analyzedfor growth as measured by OD₆₀₀ and for isoprene production as inExample 1 at 0, 1 and 3 hours. Results are shown in FIG. 47.

V. Preparation of Hydrolysate from AFEX Pretreated Corn Stover

AFEX pretreated corn stover was obtained from Michigan BiotechnologyInstitute. The pretreatment conditions were 60% moisture, 1:1 ammonialoading, and 90° C. for 30 minutes, then air dried. The moisture contentin the AFEX pretreated corn stover was 21.27%. The contents of glucanand xylan in the AFEX pretreated corn stover were 31.7% and 19.1% (drybasis), respectively. The saccharification process was as follows; 20 gof AFEX pretreated corn stover was added into a 500 ml flask with 5 mlof 1 M sodium citrate buffer pH 4.8, 2.25 ml of Accellerase 1000, 0.1 mlof Grindamyl H121 (Danisco xylanase product from Aspergillus niger forbread-making industry), and 72.65 ml of DI water. The flask was put inan orbital shaker and incubated at 50° C. for 96 hours. One sample wastaken from the shaker and analyzed using HPLC. The hydrolysate contained38.5 g/l of glucose, 21.8 g/l of xylose, and 10.3 g/l of oligomers ofglucose and/or xylose.

VI. The Effect of Yeast Extract on Isoprene Production in E. coli Grownin Fed-Batch Culture

Fermentation was performed at the 14-L scale as previously describedwith E. coli cells containing the pTrcKudzu yIDI DXS plasmid describedabove. Yeast extract (Bio Springer, Montreal, Quebec, Canada) was fed atan exponential rate. The total amount of yeast extract delivered to thefermentor was varied between 70-830 g during the 40 hour fermentation.Optical density of the fermentation broth was measured at a wavelengthof 550 nm. The final optical density within the fermentors wasproportional to the amount of yeast extract added (FIG. 48A). Theisoprene level in the off-gas from the fermentor was determined aspreviously described. The isoprene titer increased over the course ofthe fermentation (FIG. 48B). The amount of isoprene produced waslinearly proportional to the amount of fed yeast extract (FIG. 48C).

VII. Production of Isoprene in 500 L Fermentation of pTrcKudzu DXS yIDI

A 500 liter fermentation of E. coli cells with a kudzu isoprenesynthase, S. cerevisiae IDI, and E. coli DXS nucleic acids (E. coli BL21(λDE3) pTrc Kudzu dxs yidi) was used to produce isoprene. The levels ofisoprene varied from 50 to 300 μg/L over a time period of 15 hours. Onthe basis of the average isoprene concentrations, the average flowthrough the device and the extent of isoprene breakthrough, the amountof isoprene collected was calculated to be approximately 17 g.

VIII. Production of Isoprene in 500 L Fermentation of E. coli Grown inFed-Batch Culture

Medium Recipe (Per Liter Fermentation Medium):

K₂HPO₄ 7.5 g, MgSO₄*7H₂O 2 g, citric acid monohydrate 2 g, ferricammonium citrate 0.3 g, yeast extract 0.5 g, 1000× Modified Trace MetalSolution 1 ml. All of the components were added together and dissolvedin diH₂O. This solution was autoclaved. The pH was adjusted to 7.0 withammonium gas (NH₃) and q.s. to volume. Glucose 10 g, thiamine*HCl 0.1 g,and antibiotic were added after sterilization and pH adjustment.

1000× Modified Trace Metal Solution:

Citric Acids*H₂O 40 g, MnSO₄*H₂O 30 g, NaCl 10 g, FeSO₄*7H₂O 1 g,CoCl₂*6H₂O 1 g, ZnSO₄*7H₂O 1 g, CuSO₄*5H₂O 100 mg, H₃BO₃ 100 mg,NaMoO₄*2H₂O 100 mg. Each component is dissolved one at a time in DI H₂O,pH to 3.0 with HCl/NaOH, then q.s. to volume and filter sterilized with0.22 micron filter.

Fermentation was performed in a 500-L bioreactor with E. coli cellscontaining the pTrcKudzu yIDI DXS plasmid. This experiment was carriedout to monitor isoprene formation from glucose and yeast extract at thedesired fermentation pH 7.0 and temperature 30° C. An inoculum of E.coli strain taken from a frozen vial was prepared in soytone-yeastextract-glucose medium. After the inoculum grew to OD 0.15, measured at550 nm, 20 ml was used to inoculate a bioreactor containing 2.5-Lsoytone-yeast extract-glucose medium. The 2.5-L bioreactor was grown at30° C. to OD 1.0 and 2.0-L was transferred to the 500-L bioreactor.

Yeast extract (Bio Springer, Montreal, Quebec, Canada) and glucose werefed at exponential rates. The total amount of glucose and yeast extractdelivered to the bioreactor during the 50 hour fermentation was 181.2 kgand 17.6 kg, respectively. The optical density within the bioreactorover time is shown in FIG. 49A. The isoprene level in the off-gas fromthe bioreactor was determined as previously described. The isoprenetiter increased over the course of the fermentation (FIG. 49B). Thetotal amount of isoprene produced during the 50 hour fermentation was55.1 g and the time course of production is shown in FIG. 49C.

Example 8 Production of Isoprene in E. coli Expressing Kudzu IsopreneSynthase and Recombinant Mevalonic Acid Pathway Genes

I. Cloning the Lower MVA Pathway

The strategy for cloning the lower mevalonic pathway was as follows.Four genes of the mevalonic acid biosynthesis pathway; mevalonate kinase(MVK), phosphomevalonate kinase (PMK), diphosphomevalonte decarboxylase(MVD) and isopentenyl diphosphate isomerase genes were amplified by PCRfrom S. cerevisiae chromosomal DNA and cloned individually into the pCRBluntII TOPO plasmid (Invitrogen). In some cases, the idi gene wasamplified from E. coli chromosomal DNA. The primers were designed suchthat an E. coli consensus RBS (AGGAGGT (SEQ ID NO:80) or AAGGAGG (SEQ IDNO:81)) was inserted at the 5′ end, 8 by upstream of the start codon anda PstI site was added at the 3′ end. The genes were then cloned one byone into the pTrcHis2B vector until the entire pathway was assembled.

Chromosomal DNA from S. cerevisiae S288C was obtained from ATCC (ATCC204508D). The MVK gene was amplified from the chromosome of S.cerevisiae using primers MVKF(5′-AGGAGGTAAAAAAACATGTCATTACCGTTCTTAACTTCTGC, SEQ ID NO:21) andMVK-Pst1-R (5′-ATGGCTGCAGGCCTATCGCAAATTAGCTTATGAAGTCCATGGTAAATTCGTG, SEQID NO:22) using PfuTurbo as per manufacturer's instructions. The correctsized PCR product (1370 bp) was identified by electrophoresis through a1.2% E-gel (Invitrogen) and cloned into pZeroBLUNT TOPO. The resultingplasmid was designated pMVK1. The plasmid pMVK1 was digested with Sadand Taql restriction endonucleases and the fragment was gel purified andligated into pTrcHis2B digested with Sad and BstBI. The resultingplasmid was named pTrcMVK1.

The second gene in the mevalonic acid biosynthesis pathway, PMK, wasamplified by PCR using primers: PstI-PMK1 R (5′-GAATTCGCCCTTCTGCAGCTACC,SEQ ID NO:23) and BsiHKA I-PMK1 F(5′-CGACTGGTGCACCCTTAAGGAGGAAAAAAACATGTCAG, SEQ ID NO:24). The PCRreaction was performed using Pfu Turbo polymerase (Stratagene) as permanufacturer's instructions. The correct sized product (1387 bp) wasdigested with PstI and BsiHKI and ligated into pTrcMVK1 digested withPstI. The resulting plasmid was named pTrcKK. The MVD and the idi geneswere cloned in the same manner. PCR was carried out using the primerpairs PstI-MVD 1 R (5′-GTGCTGGAATTCGCCCTTCTGCAGC, SEQ ID NO:25) andNsiI-MVD 1 F (5′-GTAGATGCATGCAGAATTCGCCCTTAAGGAGG, SEQ ID NO:26) toamplify the MVD gene and PstI-YIDI 1 R (5′-CCTTCTGCAGGACGCGTTGTTATAGC,SEQ ID NO:27) and NsiI-YIDI 1 F(5′-CATCAATGCATCGCCCTTAGGAGGTAAAAAAAAATGAC, SEQ ID NO:28) to amplify theyIDI gene. In some cases the IPP isomerase gene, idi from E. coli wasused. To amplify idi from E. coli chromosomal DNA, the following primerset was used: PstI-CIDI 1 R (5′-GTGTGATGGATATCTGCAGAATTCG, SEQ ID NO:29)and NsiI-CIDI 1 F (5′-CATCAATGCATCGCCCTTAGGAGGTAAAAAAACATG, SEQ IDNO:30). Template DNA was chromosomal DNA isolated by standard methodsfrom E. coli FM5 (WO 96/35796 and WO 2004/033646, which are each herebyincorporated by reference in their entireties, particularly with respectto isolation of nucleic acids). The final plasmids were named pKKDIy forthe construct encoding the yeast idi gene or pKKDIc for the constructencoding the E. coli idi gene. The plasmids were transformed into E.coli hosts BL21 for subsequent analysis. In some cases the isoprenesynthase from kudzu was cloned into pKKDIy yielding plasmid pKKDIyIS.

The lower MVA pathway was also cloned into pTrc containing a kanamycinantibiotic resistance marker. The plasmid pTrcKKDIy was digested withrestriction endonucleases ApaI and PstI, the 5930 by fragment wasseparated on a 1.2% agarose E-gel and purified using the Qiagen GelPurification kit according to the manufacturer's instructions. Theplasmid pTrcKudzuKan, described in Example 7, was digested withrestriction endonucleases ApaI and PstI, and the 3338 by fragmentcontaining the vector was purified from a 1.2% E-gel using the QiagenGel Purification kit. The 3338 by vector fragment and the 5930 by lowerMVA pathway fragment were ligated using the Roche Quick Ligation kit.The ligation mix was transformed into E. coli TOP10 cells andtranformants were grown at 37° C. overnight with selection on LAcontaining kanamycin (50 μg/ml). The transformants were verified byrestriction enzyme digestion and one was frozen as a stock. The plasmidwas designated pTrcKanKKDIy.

II. Cloning a Kudzu Isoprene Synthase Gene into pTrcKanKKDIy

The kudzu isoprene synthase gene was amplified by PCR from pTrcKudzu,described in Example 1, using primers MCM505′-GATCATGCATTCGCCCTTAGGAGGTAAAAAAACATGTGTGCGACCTCTTCTCAA TTTACT (SEQ IDNO:31) and MCM53 5′-CGGTCGACGGATCCCTGCAGTTAGACATACATCAGCTG (SEQ IDNO:32). The resulting PCR fragment was cloned into pCR2.1 andtransformed into E. coli TOP10. This fragment contains the codingsequence for kudzu isoprene synthase and an upstream region containing aRBS from E. coli. Transformants were incubated overnight at 37° C. withselection on LA containing carbenicillin (50 μg/ml). The correctinsertion of the fragment was verified by sequencing and this strain wasdesignated MCM93. The plasmid from strain MCM93 was digested withrestriction endonucleases NsiI and PstI to liberate a 1724 by insertcontaining the RBS and kudzu isoprene synthase. The 1724 by fragment wasseparated on a 1.2% agarose E-gel and purified using the Qiagen GelPurification kit according to the manufacturer's instructions. PlasmidpTrcKanKKDIy was digested with the restriction endonuclease PstI,treated with SAP for 30 minutes at 37° C. and purified using the QiagenPCR cleanup kit. The plasmid and kudzu isoprene synthase encoding DNAfragment were ligated using the Roche Quick Ligation kit. The ligationmix was transformed into E. coli TOP10 cells and transformants weregrown overnight at 37° C. with selection on LA containing Kanamycin at50 μg/ml. The correct transformant was verified by restriction digestionand the plasmid was designated pTrcKKDyIkISKan (FIGS. 24 and 25). Thisplasmid was transformed into BL21(XDE3) cells (Invitrogen).

III. Isoprene Production from Mevalonate in E. coli Expressing theRecombinant Lower Mevalonate Pathway and Isoprene Synthase from Kudzu.

Strain BL21/pTrcKKDyIkISKan was cultured in MOPS medium (Neidhardt etal., (1974) J. Bacteriology 119:736-747) adjusted to pH 7.1 andsupplemented with 0.5% glucose and 0.5% mevalonic acid. A controlculture was also set up using identical conditions but without theaddition of 0.5% mevalonic acid. The culture was started from anovernight seed culture with a 1% inoculum and induced with 500 μM IPTGwhen the culture had reached an OD₆₀₀ of 0.3 to 0.5. The cultures weregrown at 30° C. with shaking at 250 rpm. The production of isoprene wasanalyzed 3 hours after induction by using the head space assay describedin Example 1. Maximum production of isoprene was 6.67×10⁴nmol/L_(broth)/OD₆₀₀/hr where L_(broth) is the volume of broth andincludes both the volume of the cell medium and the volume of the cells.The control culture not supplemented with mevalonic acid did not producemeasurable isoprene.

IV. Cloning the Upper MVA Pathway

The upper mevalonate biosynthetic pathway, comprising two genes encodingthree enzymatic activities, was cloned from Enterococcus faecalis. ThemvaE gene encodes a protein with the enzymatic activities of bothacetyl-CoA acetyltransferase and 3-hydroxy-3-methylglutaryl-CoA(HMG-CoA) reductase, the first and third proteins in the pathway, andthe mvaS gene encodes second enzyme in the pathway, HMG-CoA synthase.The mvaE gene was amplified from E. faecalis genomic DNA (ATCC700802D-5) with an E. coli ribosome binding site and a spacer in frontusing the following primers:

CF 07-60 (+) Start of mvaE w/ RBS + ATG start codon SacI (SEQ ID NO: 34)5′-GAGACATGAGCTCAGGAGGTAAAAAAACATGAAAACAGTAGTTAT TATTGCF 07-62 (−) Fuse mvaE to mvaS with RBS in between (SEQ ID NO: 35)5′-TTTATCAATCCCAATTGTCATGTTTTTTTACCTCCTTTATTGTTT TCTTAAATC

The mvaS gene was amplified from E. faecalis genomic DNA (ATCC700802D-5) with a RBS and spacer from E. coli in front using thefollowing primers:

CF 07-61 (+) Fuse mvaE to mvaS with RBS in between (SEQ ID NO: 36)5′-GATTTAAGAAAACAATAAAGGAGGTAAAAAAACATGACAATTGGG ATTGATAAACF 07-102 (−) End of mvaS gene BglII (SEQ ID NO: 37)5′-GACATGACATAGATCTTTAGTTTCGATAAGAACGAACGGT

The PCR fragments were fused together with PCR using the followingprimers:

CF 07-60 (+) Start of mvaE w/ RBS + ATG start codon SacI (SEQ ID NO: 34)5′-GAGACATGAGCTCAGGAGGTAAAAAAACATGAAAACAGTAGTTAT TATTGCF 07-102 (−) End of mvaS gene BglII (SEQ ID NO: 37)5′-GACATGACATAGATCTTTAGTTTCGATAAGAACGAACGGT

The fusion PCR fragment was purified using a Qiagen kit and digestedwith the restriction enzymes Sad and BglII. This digested DNA fragmentwas gel purified using a Qiagen kit and ligated into the commerciallyavailable vector pTrcHis2A, which had been digested with SacI and BglIIand gel purified.

The ligation mix was transformed into E. coli Top 10 cells and colonieswere selected on LA and 50 μg/ml carbenicillin plates. A total of sixcolonies were chosen and grown overnight in LB and 50 μg/mlcarbenicillin and plasmids were isolated using a Qiagen kit. Theplasmids were digested with Sad and BglII to check for inserts and onecorrect plasmid was sequenced with the following primers:

CF 07-58 (+) Start of mvaE gene (SEQ ID NO: 38)5′-ATGAAAACAGTAGTTATTATTGATGC CF 07-59 (−) End of mvaE gene(SEQ ID NO: 39) 5′-ATGTTATTGTTTTCTTAAATCATTTAAAATAGCCF 07-82 (+) Start of mvaS gene (SEQ ID NO: 40)5′-ATGACAATTGGGATTGATAAAATTAG CF 07-83 (−) End of mvaS gene(SEQ ID NO: 41) 5′-TTAGTTTCGATAAGAACGAACGGTCF 07-86 (+) Sequence in mvaE (SEQ ID NO: 42) 5′-GAAATAGCCCCATTAGAAGTATCCF 07-87 (+) Sequence in mvaE (SEQ ID NO: 43)5′-TTGCCAATCATATGATTGAAAATC CF 07-88 (+) Sequence in mvaE(SEQ ID NO: 44) 5′-GCTATGCTTCATTAGATCCTTATCG CF 07-89 (+) Sequence mvaS(SEQ ID NO: 45) 5′-GAAACCTACATCCAATCTTTTGCCC

The plasmid called pTrcHis2AUpperPathway#1 was correct by sequencing andwas transformed into the commercially available E. coli strain BL21.Selection was done on LA and 50 μg/ml carbenicillin. Two transformantswere chosen and grown in LB and 50 μg/ml carbenicillin until theyreached an OD₆₀₀ of 1.5. Both strains were frozen in a vial at −80° C.in the presence of glycerol. Strains were designated CF 449 forpTrcHis2AUpperPathway#1 in BL21, isolate #1 and CF 450 forpTrcHis2AUpperPathway#1 in BL21, isolate #2. Both clones were found tobehave identically when analyzed.

V. Cloning of UpperMVA Pathway into pCL1920

The plasmid pTrcHis2AUpperPathway was digested with the restrictionendonuclease Sspl to release a fragment containing pTrc-mvaE-mvaS-(Histag)-terminator. In this fragment, the his-tag was not translated. Thisblunt ended 4.5 kbp fragment was purified from a 1.2% E-gel using theQiagen Gel Purification kit. A dephosphorylated, blunt ended 4.2 kbpfragment from pCL1920 was prepared by digesting the vector with therestriction endonuclease PvuII, treating with SAP and gel purifying froma 1.2% E-gel using the Qiagen Gel Purification kit. The two fragmentswere ligated using the Roche Quick Ligation Kit and transformed intoTOP10 chemically competent cells. Transformants were selected on LAcontaining spectinomycin (50 μg/ml). A correct colony was identified byscreening for the presence of the insert by PCR. The plasmid wasdesignated pCL PtrcUpperPathway (FIGS. 26 and 27).

VI. Strains Expressing the Combined Upper and Lower Mevalonic AcidPathways

To obtain a strain with a complete mevalonic acid pathway plus kudzuisoprene synthase, plasmids pTrcKKDylklSkan and pCLpTrcUpperPathway wereboth transformed into BL21(λDE3) competent cells (Invitrogen) andtransformants were selected on LA containing kanamycin (50 μg/ml) andSpectinomycin (50 μg/ml). The transformants were checked by plasmid prepto ensure that both plasmids were retained in the host. The strain wasdesignated MCM127.

VII. Production of Mevalonic Acid from Glucose in E. coli/pUpperpathway

Single colonies of the BL21/pTrcHis2A-mvaE/mvaS or FM5/ppTrcHis2A-mvaE/mvaS are inoculated into LB and carbenicillin (100 μg/ml)and are grown overnight at 37° C. with shaking at 200 rpm. Thesecultures were diluted into 50 ml medium in 250 ml baffled flasks to anOD₆₀₀ of 0.1. The medium was TM3, 1 or 2% glucose, carbenicillin (100ug/ml) or TM3, 1% glucose. hydrolyzed soy oil, and carbenicillin (100ug/ml) or TM3 and biomass (prepared bagasse, corn stover orswitchgrass). Cultures were grown at 30° C. with shaking at 200 rpm forapproximately 2-3 hours until an OD₆₀₀ of 0.4 was reached. At this pointthe expression from the mvaE mvaS construct was induced by the additionof IPTG (400 μM). Cultures were incubated for a further 20 or 40 hourswith samples taken at 2 hour intervals to 6 hour post induction and thenat 24, 36 and 48 hours as needed. Sampling was done by removing 1 ml ofculture, measuring the OD₆₀₀, pelleting the cells in a microfuge,removing the supernatant and analyzing it for mevalonic acid.

A 14 liter fermentation of E. coli cells with nucleic acids encodingEnterococcus faecalis AA-CoA thiolase, HMG-CoA synthase, and HMG-CoAreductase polypeptides produced 22 grams of mevalonic acid with TM3medium and 2% glucose as the cell medium. A shake flask of these cellsproduced 2-4 grams of mevalonic acid per liter with LB medium and 1%glucose as the cell culture medium. The production of mevalonic acid inthese strains indicated that the MVA pathway was functional in E. coli.

VIII. Production of Isoprene from E. coli BL21 Containing the Upper andLower MVA Pathway Plus Kudzu Isoprene Synthase.

The following strains were created by transforming in variouscombinations of plasmids containing the upper and lower MVA pathway andthe kudzu isoprene synthase gene as described above and the plasmidscontaining the idi, dxs, and dxr and isoprene synthase genes describedin Example 7. The host cells used were chemically competent BL21(λDE3)and the transformations were done by standard methods. Transformantswere selected on L agar containing kanamycin (50 μg/ml) or kanamycinplus spectinomycin (both at a concentration of 50 μg/ml). Plates weregrown at 37° C. The resulting strains were designated as follows:

Grown on Kanamycin Plus Spectinomycin (50 μg/ml each)

-   MCM127—pCL Upper MVA and pTrcKKDyIkIS (kan) in BL21(λDE3)-   MCM131—pCL1920 and pTrcKKDyIkIS (kan) in BL21(λDE3)-   MCM125—pCL Upper MVA and pTrcHis2B (kan) in BL21(λDE3)    Grown on Kanamycin (50 μg/ml)-   MCM64—pTrcKudzu yIDI DXS (kan) in BL21(λDE3)-   MCM50—pTrcKudzu (kan) in BL21(λDE3)-   MCM123—pTrcKudzu yIDI DXS DXR (kan) in BL21(λDE3)

The above strains were streaked from freezer stocks to LA andappropriate antibiotic and grown overnight at 37° C. A single colonyfrom each plate was used to inoculate shake flasks (25 ml LB and theappropriate antibiotic). The flasks were incubated at 22° C. overnightwith shaking at 200 rpm. The next morning the flasks were transferred toa 37° C. incubator and grown for a further 4.5 hours with shaking at 200rpm. The 25 ml cultures were centrifuged to pellet the cells and thecells were resuspended in 5 ml LB and the appropriate antibiotic. Thecultures were then diluted into 25 ml LB, % glucose, and the appropriateantibiotic to an OD₆₀₀ of 0.1. Two flasks for each strain were set up,one set for induction with IPTG (800 μM) the second set was not induced.The cultures were incubated at 37° C. with shaking at 250 rpm. One setof the cultures were induced after 1.50 hours (immediately followingsampling time point 1). At each sampling time point, the OD₆₀₀ wasmeasured and the amount of isoprene determined as described inExample 1. Results are presented in Table 3. The amount of isoprene madeis presented as the amount at the peak production for the particularstrain.

TABLE 3 Production of isoprene in E. coli strains Strain Isoprene(μg/L_(broth)/hr/OD MCM50 23.8 MCM64 289 MCM125 ND MCM131 Trace MCM127874 ND: not detected Trace: peak present but not integrable.IX. Analysis of Mevalonic Acid

Mevalonolactone (1.0 g, 7.7 mmol) (CAS# 503-48-0) was supplied fromSigma-Aldrich (WI, USA) as a syrup that was dissolved in water (7.7 mL)and was treated with potassium hydroxide (7.7 mmol) in order to generatethe potassium salt of mevalonic acid. The conversion to mevalonic acidwas confirmed by ¹H NMR analysis. Samples for HPLC analysis wereprepared by centrifugation at 14,000 rpm for 5 minutes to remove cells,followed by the addition of a 300 μl aliquot of supernatant to 900 μl ofH₂O. Perchloric acid (36 μl of a 70% solution) was then added followedby mixing and cooling on ice for 5 minutes. The samples were thencentrifuged again (14,000 rpm for 5 min) and the supernatant transferredto HPLC. Mevalonic acid standards (20, 10, 5, 1 and 0.5 g/L) wereprepared in the same fashion. Analysis of mevalonic acid (20 uLinjection volume) was performed by HPLC using a BioRad Aminex 87-H+column (300 mm by 7.0 mm) eluted with 5 mM sulfuric acid at 0.6 mL/minwith refractive index (RI) detection. Under these conditions mevalonicacid eluted as the lactone form at 18.5 minutes.

Example 9 Construction of the Upper and Lower MVA Pathway forIntegration into Bacillus subtilis

I. Construction of the Upper MVA Pathway in Bacillus subtilis

The upper pathway from Enterococcus faecalis is integrated into B.subtilis under control of the aprE promoter. The upper pathway consistsof two genes; mvaE, which encodes for AACT and HMGR, and mvaS, whichencodes for HMGS. The two genes are fused together with a stop codon inbetween, an RBS site in front of mvaS, and are under the control of theaprE promoter. A terminator is situated after the mvaE gene. Thechloramphenicol resistance marker is cloned after the mvaE gene and theconstruct is integrated at the aprE locus by double cross over usingflanking regions of homology.

Four DNA fragments are amplified by PCR such that they contain overhangsthat will allow them to be fused together by a PCR reaction. PCRamplifications are carried out using Herculase polymerase according tomanufacturer's instructions.

1: PaprE CF 07-134 (+) Start of aprE promoter PstI (SEQ ID NO: 82)5′-GACATCTGCAGCTCCATTTTCTTCTGC CF 07-94 (−) Fuse PaprE to mvaE(SEQ ID NO: 83) 5′-CAATAATAACTACTGTTTTCACTCTTTACCCTCTCCTTTTAATemplate: Bacillus subtilis chromosomal DNA 2: mvaECF 07-93 (+) fuse mvaE to the aprE promoter (GTG start codon)(SEQ ID NO: 84) 5′-TTAAAAGGAGAGGGTAAAGAGTGAAAACAGTAGTTATTATTGCF 07-62 (−) Fuse mvaE to mvaS with RBS in between (SEQ ID NO: 35)5′-TTTATCAATCCCAATTGTCATGTTTTTTTACCTCCTTTATTGTTT TCTTAAATCTemplate: Enterococcus faecalis chromosomal DNA (from ATCC) 3. mvaSCF 07-61 (+) Fuse mvaE to mvaS with RBS in between (SEQ ID NO: 36)5′-GATTTAAGAAAACAATAAAGGAGGTAAAAAAACATGACAATTGGG ATTGATAAACF 07-124 (−) Fuse the end of mvaS to the terminator (SEQ ID NO: 85)5′-CGGGGCCAAGGCCGGTTTTTTTTAGTTTCGATAAGAACGAACGGTTemplate: Enterococcus faecalis chromosomal DNA4. B. amyliquefaciens alkaline serine protease terminatorCF 07-123 (+) Fuse the end of mvaS to the terminator (SEQ ID NO: 86)5′-ACCGTTCGTTCTTATCGAAACTAAAAAAAACCGGCCTTGGCCCCGCF 07-46 (−) End of B. amyliquefaciens terminator BamHI (SEQ ID NO: 63)5′-GACATGACGGATCCGATTACGAATGCCGTCTCTemplate: Bacillus amyliquefaciens chromosomal DNA PCR Fusion Reactions5. Fuse mvaE to mvaS CF 07-93 (+) fuse mvaE to the aprE promoter(GTG start codon) (SEQ ID NO: 84)5′-TTAAAAGGAGAGGGTAAAGAGTGAAAACAGTAGTTATTATTGCF 07-124 (−) Fuse the end of mvaS to the terminator (SEQ ID NO: 85)5′-CGGGGCCAAGGCCGGTTTTTTTTAGTTTCGATAAGAACGAACGGTTemplate: #2 and 3 from above 6. Fuse mvaE-mvaS to aprE promoterCF 07-134 (+) Start of aprE promoter PstI (SEQ ID NO: 82)5′-GACATCTGCAGCTCCATTTTCTTCTGC CF 07-124 (−) Fuse the end of mvaS to theterminator (SEQ ID NO: 85)5′-CGGGGCCAAGGCCGGTTTTTTTTAGTTTCGATAAGAACGAACGGTTemplate #1 and #4 from above 7. Fuse PaprE-mvaE-mvaS to terminatorCF 07-134 (+) Start of aprE promoter PstI (SEQ ID NO: 82)5′-GACATCTGCAGCTCCATTTTCTTCTGCCF 07-46 (−) End of B. amyliquefaciens terminator BamHI (SEQ ID NO: 63)5′-GACATGACGGATCCGATTACGAATGCCGTCTC Template: #4 and #6

The product is digested with restriction endonucleases PstI/BamHI andligated to pJM102 (Perego, M. 1993. Integrational vectors for geneticmanipulation in Bacillus subtilis, p. 615-624. In A. L. Sonenshein, J.A. Hoch, and R. Losick (ed.), Bacillus subtilis and other gram-positivebacteria: biochemistry, physiology, and molecular genetics. AmericanSociety for Microbiology, Washington, D.C.) which is digested withPstI/BamHI. The ligation is transformed into E. coli TOP 10 chemicallycompetent cells and transformants are selected on LA containingcarbenicillin (50 μg/ml). The correct plasmid is identified bysequencing and is designated pJMUpperpathway2 (FIGS. 50 and 51).Purified plasmid DNA is transformed into Bacillus subtilis aprEnprEPxyl-comK and transformants are selected on L agar containingchloramphenicol (5 μg/ml). A correct colony is selected and is platedsequentially on L agar containing chloramphenicol 10, 15 and 25 μg/ml toamplify the number of copies of the cassette containing the upperpathway.

The resulting strain is tested for mevalonic acid production by growingin LB containing 1% glucose and 1%. Cultures are analyzed by GC for theproduction of mevalonic acid.

This strain is used subsequently as a host for the integration of thelower mevalonic acid pathway.

The following primers are used to sequence the various constructs above.

Sequencing primers: CF 07-134 (+) Start of aprE promoter PstI(SEQ ID NO: 82) 5′-GACATCTGCAGCTCCATTTTCTTCTGCCF 07-58 (+) Start of mvaE gene (SEQ ID NO: 38)5′-ATGAAAACAGTAGTTATTATTGATGC CF 07-59 (−) End of mvaE gene(SEQ ID NO: 39) 5′-ATGTTATTGTTTTCTTAAATCATTTAAAATAGCCF 07-82 (+) Start of mvaS gene (SEQ ID NO: 40)5′-ATGACAATTGGGATTGATAAAATTAG CF 07-83 (−) End of mvaS gene(SEQ ID NO: 41) 5′-TTAGTTTCGATAAGAACGAACGGTCF 07-86 (+) Sequence in mvaE (SEQ ID NO: 42) 5′-GAAATAGCCCCATTAGAAGTATCCF 07-87 (+) Sequence in mvaE (SEQ ID NO: 43)5′-TTGCCAATCATATGATTGAAAATC CF 07-88 (+) Sequence in mvaE(SEQ ID NO: 44) 5′-GCTATGCTTCATTAGATCCTTATCG CF 07-89 (+) Sequence mvaS(SEQ ID NO: 45) 5′-GAAACCTACATCCAATCTTTTGCCC

Transformants are selected on LA containing chloramphenicol at aconcentration of 5 μg/ml. One colony is confirmed to have the correctintegration by sequencing and is plated on LA containing increasingconcentrations of chloramphenicol over several days, to a final level of25 μg/ml. This results in amplification of the cassette containing thegenes of interest. The resulting strain is designated CF 455:pJMupperpathway#1×Bacillus subtilis aprEnprE Pxyl comK (amplified togrow on LA containing chloramphenicol 25 μg/ml).

II. Construction of the Lower MVA Pathway in Bacillus subtilis

The lower MVA pathway, consisting of the genes mvk1, pmk, mpd and idiare combined in a cassette consisting of flanking DNA regions from thenprE region of the B. subtilis chromosome (site of integration), theaprE promoter, and the spectinomycin resistance marker (see FIGS. 28 and29). This cassette is synthesized by DNA2.0 and is integrated into thechromosome of B. subtilis containing the upper MVA pathway integrated atthe aprE locus. The kudzu isoprene synthase gene is expressed from thereplicating plasmid described in Example 4 and is transformed into thestrain with both upper and lower pathways integrated.

Example 10 Production of Isoprene in E. coli Expressing M. mazeiMevalonate Kinase and P. alba Isoprene Synthase

I. Construction of Vectors and Strains Encoding M. mazei MevalonateKinase (MVK) and P. alba Isoprene Synthase

(i) Construction of Strain EWL201 (BL21, Cm-GI1.2-KKDyI)

E. coli BL21 (Novagen brand, EMD Biosciences, Inc.) was a recipientstrain, transduced with MCM331 P1 lysate (lysate prepared according tothe method described in Ausubel, et al., Current Protocols in MolecularBiology. John Wiley and Sons, Inc.). Transductants were selected for byspreading cells onto L Agar and 20 μg/μl chloramphenicol. The plateswere incubated overnight at 30° C. Analysis of transductants showed nocolonies on control plates (water+cells control plate for reversion andwater and P1 lysate control plate for lysate contamination.

Four transductants were picked and used to inoculate 5 mL L Broth and 20μg/μl chloramphenicol. The cultures were grown overnight at 30° C. withshaking at 200 rpm. To make genomic DNA preps of each transductant forPCR analysis, 1.5 mL of overnight cell culture were centrifuged. Thecell pellet was resuspended with 400 μl Resuspension Buffer (20 mM Tris,1 mM EDTA, 50 mM NaCl, pH 7.5) and 4 μl RNase, DNase-free (Roche) wasadded. The tubes were incubated at 37° C. for 30 minutes followed by theaddition of 4 μl 10% SDS and 4 μl of 10 mg/ml Proteinase K stocksolution (Sigma-Aldrich). The tubes were incubated at 37° C. for 1 hour.The cell lysate was transferred into 2 ml Phase Lock Light Gel tubes(Eppendorf) and 200 μl each of saturated phenol pH7.9 (Ambion Inc.) andchloroform were added. The tubes were mixed well and microcentrifugedfor 5 minutes. A second extraction was done with 400 μl chloroform andthe aqueous layer was transferred to a new eppendorf tube. The genomicDNA was precipitated by the addition of 1 ml of 100% ethanol andcentrifugation for 5 minutes. The genomic DNA pellet was washed with 1ml 70% ethanol. The ethanol was removed and the genomic DNA pellet wasallowed to air dry briefly. The genomic DNA pellet was resuspended with200 μl TE.

Using Pfu Ultra II DNA polymerase (Stratagene) and 200 ng/μl of genomicDNA as template, 2 different sets of PCR reaction tubes were preparedaccording to manufacturer's protocol. For set 1, primers MCM130 and GBCm-Rev (Table 4) were used to ensure transductants were successfullyintegrated into the attTn7 locus. PCR parameters for set 1 were 95° C.for 2 minutes (first cycle only), 95° C. for 25 seconds, 55° C. for 25seconds, 72° C. for 25 seconds (repeat steps 2-4 for 28 cycles), 72° C.for 1 minute. For set 2, primers MVD For and MVD Rev (Table 4) were usedto ensure that the gi1.2-KKDyI operon integrated properly. PCRparameters for set 2 were 95° C. for 2 minutes (first cycle only), 95°C. for 25 seconds, 55° C. for 25 seconds, 72° C. for 10 seconds (repeatsteps 2-4 for 28 cycles), 72° C. for 1 minute. Analysis of PCR ampliconson a 1.2% E-gel (Invitrogen Corp.) showed that all 4 transductant cloneswere correct (picked one and designated as strain EWL201).

ii) Construction of Strain EWL204 (BL21, loopout-GI1.2-KKDyI)

The chloramphenicol marker was looped out of strain EWL201 using plasmidpCP20 as described by Datsenko and Wanner (2000) (Datsenko et al., ProcNatl. Acad. Sci. USA 97:6640-6645, 2000). One-step inactivation ofchromosomal genes in Escherichia coli K-12 using PCR products. (Datsenkoet al., PNAS, 97: 6640-6645, 2000). EWL201 cells were grown in L Brothto midlog phase and then washed three times in ice-cold, sterile water.An aliquot of 50 μl of cell suspension was mixed with 1 μl of pCP20 andthe cell suspension mixture was electroporated in a 2 mm cuvette(Invitrogen Corp.) at 2.5 Volts and 25 uFd using a Gene PulserElectroporator (Bio-Rad Inc.). 1 ml of LB was immediately added to thecells, then transferred to a 14 ml polypropylene tube (Sarstedt) with ametal cap.

Cells were allowed to recover by growing for 1 hour at 30° C.Transformants were selected on L Agar and 20 μg/μl chloramphenicol and50 μg/μl carbenicillin and incubated at 30° C. overnight. The next day,a single clone was grown in 10 ml L Broth and 50 μg/μl carbenicillin at30° C. until early log phase. The temperature of the growing culture wasthen shifted to 42° C. for 2 hours. Serial dilutions were made, thecells were then spread onto LA plates (no antibiotic selection), andincubated overnight at 30° C. The next day, 20 colonies were picked andpatched onto L Agar (no antibiotics) and LA and 20 μg/μl chloramphenicolplates. Plates were then incubated overnight at 30° C. Cells able togrow on LA plates, but not LA and 20 μg/μl chloramphenicol plates, weredeemed to have the chloramphenicol marker looped out (picked one anddesignated as strain EWL204).

iii) Construction of Plasmid pewl230 (pTrc P. alba)

Generation of a synthetic gene encoding Populus alba isoprene synthase(P. alba HGS) was outsourced to DNA2.0 Inc. (Menlo Park, Calif.) basedon their codon optimization method for E. coli expression. The syntheticgene was custom cloned into plasmid pET24a (Novagen brand, EMDBiosciences, Inc.) and delivered lyophilized (FIGS. 54, 55A and 55B).

A PCR reaction was performed to amplify the P. alba isoprene synthase(P. alba HGS) gene using pET24 P. alba HGS as the template, primersMCM182 and MCM192, and Herculase II Fusion DNA polymerase (Stratagene)according to manufacturer's protocol. PCR conditions were as follows:95° C. for 2 minutes (first cycle only), 95° C. for 25 seconds, 55° C.for 20 seconds, 72° C. for 1 minute, repeat for 25 cycles, with finalextension at 72° C. for 3 minutes. The P. alba isoprene synthase PCRproduct was purified using QIAquick PCR Purification Kit (Qiagen Inc.).

P. alba isoprene synthase PCR product was then digested in a 20 μlreaction containing 1 μl BspHI endonuclease (New England Biolabs) with 2μl 10×NEB Buffer 4. The reaction was incubated for 2 hours at 37° C. Thedigested PCR fragment was then purified using the QIAquick PCRPurification Kit. A secondary restriction digest was performed in a 20μl reaction containing 1 μl PstI endonuclease (Roche) with 2 μl 10×Buffer H. The reaction was incubated for 2 hours at 37° C. The digestedPCR fragment was then purified using the QIAquick PCR Purification Kit.Plasmid pTrcHis2B (Invitrogen Corp.) was digested in a 20 μl reactioncontaining 1 μl NcoI endonuclease (Roche), 1 μl PstI endonuclease, and 2μl 10× Buffer H. The reaction was incubated for 2 hours at 37° C. Thedigested pTrcHis2B vector was gel purified using a 1.2% E-gel(Invitrogen Corp.) and extracted using the QIAquick Gel Extraction Kit(Qiagen) (FIG. 56). Using the compatible cohesive ends of BspHI and NcoIsites, a 20 μl ligation reaction was prepared containing 5 μl P. albaisoprene synthase insert, 2 μl pTrc vector, 1 μl T4 DNA ligase (NewEngland Biolabs), 2 μl 10× ligase buffer, and 10 μl ddH₂O. The ligationmixture was incubated at room temperature for 40 minutes. The ligationmixture was desalted by floating a 0.025 μm nitrocellulose membranefilter (Millipore) in a petri dish of ddH₂O and applying the ligationmixture gently on top of the nitrocellulose membrane filter for 30minutes at room temperature. MCM446 cells (See section II) were grown inLB to midlog phase and then washed three times in ice-cold, sterilewater. An aliquot of 50 μl of cell suspension was mixed with 5 μl ofdesalted pTrc P. alba HGS ligation mix. The cell suspension mixture waselectroporated in a 2 mm cuvette at 2.5 Volts and 25 uFd using a GenePulser Electroporator. 1 ml of LB is immediately added to the cells,then transferred to a 14 ml polypropylene tube (Sarstedt) with a metalcap.

Cells were allowed to recover by growing for 2 hour at 30° C.Transformants were selected on L Agar and 50 μg/μl carbenicillin and 10mM mevalonic acid and incubated at 30° C. The next day, 6 transformantswere picked and grown in 5 ml L Broth and 50 μg/μl carbenicillin tubesovernight at 30° C. Plasmid preps were performed on the overnightcultures using QIAquick Spin Miniprep Kit (Qiagen). Due to the use ofBL21 cells for propagating plasmids, a modification of washing the spincolumns with PB Buffer 5× and PE Buffer 3× was incorporated to thestandard manufacturer's protocol for achieving high quality plasmid DNA.Plasmids were digested with PstI in a 20 μl reaction to ensure thecorrect sized linear fragment. All 6 plasmids were the correct size andshipped to Quintara Biosciences (Berkeley, Calif.) for sequencing withprimers MCM65, MCM66, EL1000 (Table 4). DNA sequencing results showedall 6 plasmids were correct. Picked one and designated plasmid as EWL230(FIGS. 57, 58A and 58B).

iv) Construction of Plasmid pEWL244 (pTrc P. alba-mMVK)

A PCR reaction was performed to amplify the Methanosarcina mazei (M.mazei) MVK gene using MCM376 as the template (see section v), primersMCM165 and MCM177 (see Table 4), and Pfu Ultra II Fusion DNA polymerase(Stratagene) according to manufacturer's protocol. PCR conditions wereas follows: 95° C. for 2 minutes (first cycle only), 95° C. for 25seconds, 55° C. for 25 seconds, 72° C. for 18 seconds, repeat for 28cycles, with final extension at 72° C. for 1 minute. The M. mazei MVKPCR product was purified using QIAquick PCR Purification Kit (QiagenInc.).

The M. mazei MVK PCR product was then digested in a 40 μl reactioncontaining 8 μl PCR product, 2 μl Pmel endonuclease (New EnglandBiolabs), 4 μl 10×NEB Buffer 4, 4 μl 10×NEB BSA, and 22 μl of ddH₂O. Thereaction was incubated for 3 hours at 37° C. The digested PCR fragmentwas then purified using the QIAquick PCR Purification Kit. A secondaryrestriction digest was performed in a 47 μl reaction containing 2 μlNsiI endonuclease (Roche), 4.7 μl 10× Buffer H, and 40 μl of PmeIdigested M. mazei MVK fragment. The reaction was incubated for 3 hoursat 37° C. The digested PCR fragment was then gel purified using a 1.2%E-gel and extracted using the QIAquick Gel Extraction Kit. PlasmidEWL230 was digested in a 40 μl reaction containing 10 μl plasmid, 2 μlPmeI endonuclease, 4 μl 10×NEB Buffer 4, 4 μl 10×NEB BSA, and 20 μl ofddH₂O. The reaction was incubated for 3 hours at 37° C. The digested PCRfragment was then purified using the QIAquick PCR Purification Kit. Asecondary restriction digest was performed in a 47 μl reactioncontaining 2 μl PstI endonuclease, 4.7 μl 10× Buffer H, and 40 μl ofPmeI digested EWL230 linear fragment. The reaction was incubated for 3hours at 37° C. The digested PCR fragment was then gel purified using a1.2% E-gel and extracted using the QIAquick Gel Extraction Kit (FIG.59). Using the compatible cohesive ends of NsiI and PstI sites, a 20 μlligation reaction was prepared containing 8 μl M. mazei MVK insert, 3 μlEWL230 plasmid, 1 μl T4 DNA ligase, 2 μl 10× ligase buffer, and 4 μlddH₂O. The ligation mixture was incubated at overnight at 16° C. Thenext day, the ligation mixture was desalted by floating a 0.024 μmnitrocellulose membrane filter in a petri dish of ddH₂O and applying theligation mixture gently on top of the nitrocellulose membrane filter for30 minutes at room temperature. MCM446 cells were grown in LB to midlogphase and then washed three times in ice-cold, sterile water. An aliquotof 50 μl of cell suspension was mixed with 5 μl of desalted pTrc P.alba-mMVK ligation mix. The cell suspension mixture was electroporatedin a 2 mm cuvette at 2.5 Volts and 25 uFd using a Gene PulserElectroporator. 1 ml of LB is immediately added to the cells, then thecells are transferred to a 14 ml polypropylene tube with a metal cap.Cells were allowed to recover by growing for 2 hour at 30° C.Transformants were selected on LA and 50 μg/μl carbenicillin and 5 mMmevalonic acid plates and incubated at 30° C. The next day, 6transformants were picked and grown in 5 ml LB and 50 μg/μlcarbenicillin tubes overnight at 30° C. Plasmid preps were performed onthe overnight cultures using QIAquick Spin Miniprep Kit. Due to the useof BL21 cells for propagating plasmids, a modification of washing thespin columns with PB Buffer 5× and PE Buffer 3× was incorporated to thestandard manufacturer's protocol for achieving high quality plasmid DNA.Plasmids were digested with PstI in a 20 μl reaction to ensure thecorrect sized linear fragment. Three of the 6 plasmids were the correctsize and shipped to Quintara Biosciences for sequencing with primersMCM65, MCM66, EL1000, EL1003, and EL1006 (Table 4). DNA sequencingresults showed all 3 plasmids were correct. Picked one and designatedplasmid as EWL244 (FIGS. 60 and 61A-B).

v) Construction of Plasmid MCM376—MVK from M. mazei Archaeal Lower inpET200D.

The MVK ORF from the M. mazei archaeal Lower Pathway operon (FIGS.73A-C) was PCR amplified using primers MCM161 and MCM162 (Table 4) usingthe Invitrogen Platinum HiFi PCR mix. 45 uL of PCR mix was combined with1 uL template, 1 uL of each primer at 10 uM, and 2 uL water. Thereaction was cycled as follows: 94° C. for 2:00 minutes; 30 cycles of94° C. for 0:30 minutes, 55° C. for 0:30 minutes and 68° C. for 1:15minutes; and then 72° C. for 7:00 minutes, and 4° C. until cool. 3 uL ofthis PCR reaction was ligated to Invitrogen pET200D plasmid according tothe manufacturer's protocol. 3 uL of this ligation was introduced intoInvitrogen TOP10 cells, and transformants were selected on LA/kan50. Aplasmid from a transformant was isolated and the insert sequenced,resulting in MCM376 (FIGS. 74A-C).

vi) Construction of Strain EWL251 (BL21(DE3), Cm-GI1.2-KKDyI, pTrc P.alba-mMVK)

MCM331 cells (which contain chromosomal construct gi1.2KKDyI encoding S.cerevisiae mevalonate kinase, mevalonate phosphate kinase, mevalonatepyrophosphate decarboxylase, and IPP isomerase) were grown in LB tomidlog phase and then washed three times in ice-cold, sterile water.Mixed 50 μl of cell suspension with 1 μl of plasmid EWL244. The cellsuspension mixture was electroporated in a 2 mm cuvette at 2.5 Volts and25 uFd using a Gene Pulser Electroporator. 1 ml of LB is immediatelyadded to the cells, then the cells were transferred to a 14 mlpolypropylene tube with a metal cap. Cells were allowed to recover bygrowing for 2 hours at 30° C. Transformants were selected on LA and 50μg/μl carbenicillin and 5 mM mevalonic acid plates and incubated at 37°C. One colony was selected and designated as strain EWL251.

vii) Construction of Strain EWL256 (BL21(DE3), pTrc P. alba-mMVK, pCLUpper MVA)

EWL251 cells were grown in LB to midlog phase and then washed threetimes in ice-cold, sterile water. Mixed 50 μl of cell suspension with 1μl of plasmid MCM82 (which is pCL PtrcUpperPathway encoding E. faecalismvaE and mvaS). The cell suspension mixture was electroporated in a 2 mmcuvette at 2.5 Volts and 25 uFd using a Gene Pulser Electroporator. 1 mlof LB was immediately added to the cells, then transferred to a 14 mlpolypropylene tube with a metal cap. Cells were allowed to recover bygrowing for 2 hour at 30° C. Transformants were selected on LA and 50μg/μl carbenicillin and 50 μg/μl spectinomycin plates and incubated at37° C. Picked one colony and designated as strain EWL256.

TABLE 4 Primer Sequences Primer name Primer sequence MCM130ACCAATTGCACCCGGCAGA (SEQ ID NO: 94) GB Cm GCTAAAGCGCATGCTCCAGAC Rev(SEQ ID NO: 95) MVD For GACTGGCCTCAGATGAAAGC (SEQ ID NO: 96) MVDCAAACATGTGGCATGGAAAG Rev (SEQ ID NO: 97) MCM182GGGCCCGTTTAAACTTTAACTAGACTCTGCAGTTAGCGTT CAAACGGCAGAA (SEQ ID NO: 98)MCM192 CGCATGCATGTCATGAGATGTAGCGTGTCCACCGAAAA (SEQ ID NO: 99 MCM65ACAATTTCACACAGGAAACAGC (SEQ ID NO: 100) MCM66 CCAGGCAAATTCTGTTTTATCAG(SEQ ID NO: 101) EL1000 GCACTGTCTTTCCGTCTGCTGC (SEQ ID NO: 102) MCM165GCGAACGATGCATAAAGGAGGTAAAAAAACATGGTATCCT GTTCTGCGCCGGGTAAGATTTACCTG(SEQ ID NO: 103) MCM177 GGGCCCGTTTAAACTTTAACTAGACTTTAATCTACTTTCAGACCTTGC (SEQ ID NO: 104) EL1003 GATAGTAACGGCTGCGCTGCTACC(SEQ ID NO: 105) EL1006 GACAGCTTATCATCGACTGCACG (SEQ ID NO: 106) MCM161CACCATGGTATCCTGTTCTGCG (SEQ ID NO: 107) MCM162 TTAATCTACTTTCAGACCTTGC(SEQ ID NO: 108)II. Construction of MCM442-449: BL21 and BL21(DE3) withFRT-cmR-FRT-gi1.x-mKKDyIi) Construction of Template for Recombination

FRT-based recombination cassettes, and plasmids for Red/ET-mediatedintegration and antibiotic marker loopout were obtained from GeneBridges GmbH (Germany). Procedures using these materials were carriedout according to Gene Bridges protocols. Primers MCM193 and MCM195 wereused to amplify the resistance cassette from the FRT-gb2-Cm-FRT templateusing Stratagene Herculase II Fusion kit according to the manufacturer'sprotocol. The 50 uL reaction was cycled as follows: 95° C., 2 minutes;(95° C., 20 seconds, 55° C., 20 seconds, 72° C., 1 minute)×5, (95° C.,20 seconds, 60° C., 20 seconds, 72° C., 1 minute)×25; 72° C., 3 minutes;4° C. until cool. The amplicon was purified by a Qiagen PCR columnaccording to the manufacturer's protocol and eluted in 30 uL EB (ElutionBuffer). DNA was digested with NdeI and PciI in a 20 uL reaction with 1×Roche H buffer and 0.5 uL BSA. Plasmid MCM376 was digested in a 10 uLreaction containing 1 uL each of N del, N col, and Roche H buffer.Reactions proceeded overnight at 37° C., and then cut DNA was purifiedon Qiagen PCR columns and eluted in 30 uL EB. The PCR product wasligated into MCM376 in a reaction containing 1 uL vector, 3 uL PCRproduct, 1 uL Roche Quick Ligase Buffer 2, 5 uL Buffer1, and 1 uLLigase. The reaction proceeded at room temperature for 3 hours and then5 uL was transformed into Invitrogen TOP10 cells according to themanufacturer's protocol. Transformants were selected on L agar (LA) andchloramphenicol (10 ug/mLO) at 37° C. overnight.

Transformant colonies were patched onto LA containing chloramphenicol(30 ug/mL) and kanamycin (50 ug/ml) for storage and sent to Quintara(Berkeley, Calif.) for sequencing. Four clones, one each with the fourdifferent nucleotides at the “N” in primer MCM195, were found to havethe correct sequence for the inserted promoter. Clones were grown in 5mL LB containing chloramphenicol (30 ug/mL) and kanamycin (50 ug/mL) andused for the preparation of plasmid DNA. This plasmid was retransformedinto TOP10 cells and strains were frozen as:

TABLE 5 MCM484-487 MCM484 cmR-gi1.6-MVK(mazei) in pET (clone A1-3,variable nt A) MCM485 cmR-gi1.0-MVK(mazei) in pET (clone B4-6, variablent C) MCM486 cmR-gi1.2-MVK(mazei) in pET (clone C1-5, variable nt G)MCM487 cmR-gi1.5-MVK(mazei) in pET (clone C3-3, variable nt T)ii) Creation of Recombination Target Strains MCM349 and MCM441

The chloramphenicol resistance (cmR) marker was looped out of strainMCM331 using plasmid pGB706 (GeneBridges) according to Manufacturer'sinstructions. MCM331 cells were grown to mid-log in LB and washed threetimes in iced, sterile water. A 1 uL aliquot of pGB706 DNA was added to50 uL of cell suspension and this mixture was electroporated in a 2 mmcuvette at 2.5 volts, 25 uFd followed immediately by recovery in 500 uLLB for one hour at 30 C. Transformants were selected on LB containingtetracycline (5 ug/ml) at 30° C. The following day, a clone was grown upat 30° C. in LB containing tetracycline (5 ug/ml) until visibly turbid(OD600˜0.5-0.8). This culture was streaked onto LB and grown overnightat 37° C. A clone that was unable to grow on LB containingchloramphenicol (10 ug/mL) or LB containing tetracycline (5 ug/mL) wasfrozen as MCM348. Plasmid MCM356 (pRedET carbencillin; GeneBridges) waselectroporated in as described above and transformants were selected onLB containing carbenicillin (50 ug/mL) at 30° C. A clone was grown in LBcarbenicillin (50 ug/mL) at 30° C. and frozen as MCM349.

Strain MCM441 was created by electrotransforming plasmid MCM356 intoEWL204 as above.

iii) Recombination of FRT-cmR-FRT-gi1.x-mMVK into MCM349 and MCM441

Plasmids MCM484-487 were used as template for PCR amplification withprimers MCM120 and MCM196 and Herculase II Fusion kit, according to themanufacturer's protocol. Three reactions per template were carried out,with 0, 1, or 3 uL DMSO. The 50 uL reactions were cycled as follows: 95°C., 2 minutes; (95° C., 20 seconds; 55° C. 20 seconds; 72° C., 1.5minutes) for five cycles; (95° C., 20 seconds; 60° C. 20 seconds; 72°C., 1.5 minutes) for 25 cycles; 72° C. for 3 minutes; 4° C., overnight.The three reactions from a given template were pooled and purified onQiagen PCR columns and eluted with 30 uL EB at 60° C. 5 uL DNA wasdigested with 1 uL Dpnl in 1× Roche Buffer A for 3 hours at 37° C. ThisDNA was then microdialyzed against excess water for 30 minutes.

Strains were grown in 5 mL LB containing carbenicillin (50 ug/mL) fromfresh streaks at 30 C to an OD600 of ˜0.5. 40 mM L-arabinose was addedand cultures were incubated at 37 C for 1.5 hours. Cells were harvestedand electroporated with 3 uL dialyzed amplicons above, and thenrecovered in 500 uL SOC at 37 C for 1.5-3 hours. Transformants wereselected on LA plates containing chloramphenicol (5 ug/mL) at 37° C.

Kanamycin sensitive clones were screened by PCR for insertion of theamplicon. PCR products from positive clones were sequenced to verify thesequence of inserted DNA. Amplicons were consistent with theFRT-gi1.2-yKKDyI at attTn7 in MCM441 and 348 being replaced byFRT-cmR-FRT-gi1.x-mKKDyI (The yK and mK designations refer to themevalonate kinase from Saccharomyces cerevisiae and Methanosarcina mazeirespectively).

TABLE 6A The following strains were grown in LB containingchloramphenicol (5 ug/mL) and frozen. Recombi- nation Strain Amplicon IDName Parent Template MCM442 BL21(DE3) cmR-gi1.6mKKDyI A1, MCM349 MCM484clone37 (A) MCM443 BL21(DE3) cmR-gi1.0mKKDyI B4, MCM349 MCM485 clone27(C) MCM444 BL21(DE3) cmR-gi1.2mKKDyI C1, MCM349 MCM486 clone16 (G)MCM445 BL21(DE3) cmR-gi1.5mKKDyI C3, MCM349 MCM487 clone7 (T) MCM446BL21 cmR-gi1.6mKKDyI A1-3 (A) MCM441 MCM484 MCM447 BL21 cmR-gi1.0mKKDyIB4-6 (C) MCM441 MCM485 MCM448 BL21 cmR-gi1.2mKKDyI C1-5 (G) MCM441MCM486 MCM449 BL21 cmR-gi1.5mKKDyI C3-3 (T) MCM441 MCM487

TABLE 6B Primers MCM120 AAAGTAGCCGAAGATGACGGTTTGTCACATGGAGTTGGCAGGATGTTTGATTAAAAGCAATTAACCCTCACTAAAGGGCGG (SEQ ID NO: 109) MCM193GATATACATATGAATTAACCCTCACTAAAGG (SEQ ID NO: 110) MCM195GCATGCATGACATGTTTTTTTACCTCCTTTGTTATCCGCTCACAATTAGTGGTTGAATTATTTGCTCAGGATGTGGCATNGTCAAGGGCGCGGCCGCGATCTAATACGACTCACTATAGGGCTCG (SEQ ID NO: 111) MCM196AGGCTCTCAACTCTGACATGTTTTTTTCCTCCTTAAGGGTGCAGGCCTATCGCAAATTAGCTTAATCTACTTTCAGACCTTGCT CGG (SEQ ID NO: 112)III. The Effect of Yeast Extract on Isoprene Production in E. coliExpressing Genes from the Mevalonic Acid Pathway and Grown in Fed-BatchCulture at the 15-L ScaleMedium Recipe (Per Liter Fermentation Medium):

K₂HPO₄ 7.5 g, MgSO₄*7H₂O 2 g, citric acid monohydrate 2 g, ferricammonium citrate 0.3 g, yeast extract 0.5 g, 1000× Modified Trace MetalSolution 1 ml. All of the components were added together and dissolvedin diH₂O. This solution was autoclaved. The pH was adjusted to 7.0 withammonium hydroxide (30%) and q.s. to volume. Glucose 10 g, thiamine*HCl0.1 g, and antibiotics were added after sterilization and pH adjustment.

1000× Modified Trace Metal Solution:

Citric Acids*H₂O 40 g, MnSO₄*H₂O 30 g, NaCl 10 g, FeSO₄*7H₂O 1 g,CoCl₂*6H₂O 1 g, ZnSO₄*7H₂O 1 g, CuSO₄*5H₂O 100 mg, H₃BO₃ 100 mg,NaMoO₄*2H₂O 100 mg. Each component is dissolved one at a time in Di H₂O,pH to 3.0 with HCl/NaOH, then q.s. to volume and filter sterilized witha 0.22 micron filter.

Fermentation was performed in a 15-L bioreactor with BL21 (DE3) E. colicells containing the upper mevalonic acid (MVA) pathway (pCL Upper), theintegrated lower MVA pathway (gi1.2KKDyl), and high expression ofmevalonate kinase from M. mazei and isoprene synthase from P. alba(pTrcAlba-mMVK). This experiment was carried out to monitor isopreneformation from glucose at the desired fermentation pH 7.0 andtemperature 30° C. A frozen vial of the E. coli strain was thawed andinoculated into tryptone-yeast extract medium. After the inoculum grewto OD 1.0, measured at 550 nm, 500 mL was used to inoculate a 15-Lbioreactor bringing the initial volume to 5-L.

i) Production of Isoprene in E. coli Cells (EL256) Grown in Fed-BatchCulture without Yeast Extract Feeding

Glucose was fed at an exponential rate until cells reached thestationary phase. After this time the glucose feed was decreased to meetmetabolic demands. The total amount of glucose delivered to thebioreactor during the 67 hour fermentation was 3.9 kg.

Induction was achieved by addingisopropyl-beta-D-1-thiogalactopyranoside (IPTG). The IPTG concentrationwas brought to 102 uM when the optical density at 550 nm (OD₅₅₀) reacheda value of 9. The IPTG concentration was raised to 192 uM when OD₅₅₀reached 140. The OD₅₅₀ profile within the bioreactor over time is shownin FIG. 67A. The isoprene level in the off gas from the bioreactor wasdetermined using a Hiden mass spectrometer. The isoprene titer increasedover the course of the fermentation to a final value of 35.6 g/L (FIG.67B). The total amount of isoprene produced during the 67 hourfermentation was 320.6 g and the time course of production is shown inFIG. 67C. The metabolic activity profile, as measured by TCER, is shownin FIG. 67D. The molar yield of utilized carbon that went into producingisoprene during fermentation was 17.9%. The weight percent yield ofisoprene from glucose was 8.1%.

Production of Isoprene in E. coli Cells (EL256) Grown in Fed-BatchCulture with Yeast Extract Feeding

Glucose was fed at an exponential rate until cells reached thestationary phase. After this time the glucose feed was decreased to meetmetabolic demands. The total amount of glucose delivered to thebioreactor during the 68 hour fermentation was 7.1 kg. A total of 1.06kg of yeast extract was also fed during the fermentation. Induction wasachieved by adding IPTG. The IPTG concentration was brought to 208 uMwhen the optical density at 550 nm (OD₅₅₀) reached a value of 7. TheIPTG concentration was raised to 193 uM when OD₅₅₀ reached 180. TheOD₅₅₀ profile within the bioreactor over time is shown in FIG. 68A. Theisoprene level in the off gas from the bioreactor was determined using aHiden mass spectrometer. The isoprene titer increased over the course ofthe fermentation to a maximum value of 32.2 g/L (FIG. 68B). The totalamount of isoprene produced during the 68 hour fermentation was 395.5 gand the time course of production is shown in FIG. 68C. The time courseof volumetric productivity is shown in FIG. 68D and shows that anaverage rate of 1.1 g/L/hr was maintained for between 23 and 63 hours.The metabolic activity profile, as measured by CER, is shown in FIG. 68EThe molar yield of utilized carbon that went into producing isopreneduring fermentation was 10.3%. The weight percent yield of isoprene fromglucose was 5.2%.

IV. Production of Isoprene from Different Carbon Sources in E. coliHarboring the Mevalonic Acid (MVA) Pathway and Isoprene Synthase(EWL256)

Media Recipe (Per Liter Fermentation Media):

K₂HPO₄ 13.6 g, KH₂PO₄ 13.6 g, MgSO₄*7H₂O 2 g, citric acid monohydrate 2g, ferric ammonium citrate 0.3 g, (NH₄)₂SO₄ 3.2 g, yeast extract 0.2 g,1000× Modified Trace Metal Solution 1 ml. All of the components weredissolved sequentially in diH₂O. The pH was adjusted to 6.8 withammonium hydroxide (30%) and brought to volume. Media was filtersterilized with a 0.22 micron filter. Carbon source was added to a finalconcentration of 1%. Required antibiotics were added after sterilizationand pH adjustment.

1000× Trace Metal Solution (Per Liter Fermentation Media):

Citric Acids*H₂O 40 g, MnSO₄*H₂O 30 g, NaCl 10 g, FeSO₄*7H₂O 1 g,CoCl₂*6H₂O 1 g, ZnSO₄*7H₂O 1 g, CuSO₄*5H₂O 100 mg, H₃BO₃ 100 mg,NaMoO₄*2H₂O 100 mg. Each component was dissolved one at a time in diH₂O,pH to 3.0 with HCl/NaO H, and then brought to volume and filtersterilized with a 0.22 micron filter.

i) Preparation of AFEX Biomass Hydrolysate

AFEX pretreated corn stover was hydrolyzed to prepare biomasshydrolysate containing both xylose, glucose and acetate.

AFEX pretreated corn stover, received from Michigan BiotechnologyInstitute, was used. The pretreatment conditions were, 60% moisture, 1:1ammonia loading, and 90° C. for 30 minutes, then air dried. The moisturecontent in the AFEX pretreated corn stover was 21.27%. Content of glucanand xylan in the AFEX pretreated corn stover were 31.7% and 19.1% (drybasis) respectively. The enzyme used was accellerase 1000, GrindamylH121 (Danisco xylanase product from Aspergillus niger for bread-makingindustry).

For saccharification, 20 g of AFEX pretreated corn stover was added intoa 500 ml flask, together with 5 ml of 1 M pH 4.8 sodium citrate buffer,2.25 ml of Accellerase 1000, 0.1 ml of Grindamyl H121, and 72.65 ml ofDI water. The flask was put in an orbital shaker, and incubated at 50°C. for 96 hours.

For analysis, one sample was taken from the shaker, and analyzed usingHPLC. The hydrolysate contained 37.2 g/l of glucose and 24.3 g/L ofxylose, and 7.6 g/L of oligomers of glucose and/or xylose. Additionally,the hydrolysate also contains 1.17 g/L acetate.

ii) Experimental Procedure

An inoculum of the E. coli strain EWL256 containing the MVA pathway andisoprene synthase was taken from a frozen vial and streaked onto an LBbroth agar plate containing spectinomycin (50 ug/mL) and carbinicllin(50 ug/mL) and incubated at 30° C. overnight. A single colony wasinoculated into TM3 media containing glucose, xylose, glycerol, acetateor biomass as only carbon source and grown overnight at 30° C. Cellsgrow on acetate reached a significantly lower optical density. Cellsgrown on glucose, glycerol, biomass hydrolysate or acetate were dilutedinto 20 mL of TM3 media containing the respective carbon sources toreach an optical density of between 0.1 measured at 600 nM. A negativecontrol not containing any carbon source was prepared from the glucoseovernight culture. A separate experiment was performed with glucose andxylose, where the cultures were diluted to an optical density of 0.05.All culture conditions (except for acetate and glycerol) were tested induplicates and the presented results are averaged between thesecultures. Production of isoprene was induced with 200 μM IPTG from thebeginning of the experiment. The flasks were incubated at 30° C. in anorbital shaker (200 rpm) and growth was followed by measuring opticaldensity. After the glucose fed cultures had reached an optical densityof approximately 0.4, samples were analyzed for isoprene production fromall the tested carbon sources every hour for three hours. Samples of 100μL were transferred in duplicates to 2 mL glass vials, sealed andincubated for 30 min at 30° C. The bacteria were then heat killed byincubation at 80° C. for 8 minutes. The amount of produced isoprene wasmeasured using GC-MS and specific productivity (μg/L*hr) was calculated.

iii) Results

Significant production of isoprene could be demonstrated during growthon all the tested carbon sources. These carbon sources are examples ofcommon alcohols, organic acids, sugars containing 5 or 6 carbon units(C₅ or C₆), and biomass hydrolysate.

The initial growth rate on biomass hydrolysate was comparable to thegrowth rate on glucose (FIG. 69A). The initial specific productivityduring growth on biomass hydrolysate was significantly higher thanduring growth on glucose. This demonstrates that biomass hydrolysate canbe used as an efficient source of carbon for the production of isoprene.The specific productivity declined after 255 minutes of growth onbiomass hydrolysate (FIG. 69B). The bacteria had a slower growth ratewith xylose as only carbon source when compared to glucose (FIG. 69C),but a significant specific isoprene productivity was measured (FIG.69D). This shows that both C₅ and C₆ sugars can be utilized for theproduction of isoprene via the mevalonate acid pathway.

Surprisingly, bacteria grown on acetate as the only carbon source had aspecific productivity of isoprene approximately twice as high as duringgrowth on glucose (FIG. 69A). The bacteria grew slower on acetate whencompared to glucose (FIG. 69B), but the performed experimentdemonstrates that acetate can also be used as a carbon source for theproduction of isoprene. Acetate was also present in the biomasshydrolysate as measured by HPLC.

The bacteria grew well with glycerol as only carbon source (FIG. 69A)and significant production of isoprene was demonstrated (FIG. 69B). Thisshows that common alcohols may also be used as carbon sources forproduction of isoprene via the mevalonate acid pathway.

Example 11 Expression of Isoprene-synthase from Plant in Streptomycessp.

The gene for isoprene synthase Kudzu was obtained from plasmidpJ201:19813. Plasmid pJ201:19813 encodes isoprene synthase from Pueraialobata (Kudzu plant) and was codon-optimized for Pseudomonasfluorescens, Pseudomonas putida, Rhodopseudomonas palustris andCorynebacterium (FIGS. 79A-79C (SEQ ID NO:123)). Digestion of plasmidpJ201:19813 with restriction enzymes NdeI and BamHI liberated geneiso19813 that was ligated into the Streptomyces-E. coli shuttle vectorpUWL201PW (Doumith et al., Mol. Gen. Genet. 264: 477-485, 2000; FIG. 71)to generate pUWL201_iso. Successful cloning was verified by restrictionanalysis of pUWL201_iso. Expression of isoprene synthase iso19813 wasunder control of the erm-promoter which allows for constitutiveexpression in Streptomycetes species, but not for expression in E. coli.

PUWL201PW (no insert) and pUWL201_iso were introduced in Streptomycesalbus J1074 (Sanchez et al., Chem. Biol. 9:519-531, 2002) bytransformation of protoplasts as described by Hopwood et al., The Johninnes foundation, Norwich, 1985.

A 200 μl aliquot of protoplast suspensions was transformed with 1.9 μgpUWL201PW or 2.9 μg pUWL201_iso. After incubation overnight at 28° C. onnon-selective R5-agarplates, positive transformants were selected byfurther incubation for 4 days in R3-overlay agar containing thiostrepton(250 μg/ml). Thiostrepton resistant transformants were examined forpresence of the pUWL-plasmids by plasmid preparation using Plasmid MiniKit (Qiagen). Prepared plasmid DNA was reintroduced in E. coli DH5a togenerate sufficient amounts of plasmid DNA to be analyzed by restrictionanalysis. Positive transformants were selected on ampicillin-containingL-agar plates and insert analysis was done by digestion of plasmid DNAwith NdeI and BamHI endonucleases. Isoprene synthase was identified as a1.7 kb fragment in positive pUWL201 iso clones while in the controlstrains (pUWL201PW) no such fragment was observed.

Wild type strain and transformants of S. albus containing controlplasmid pUWL201PW or isoprene synthase encoding pUWL201_iso wereanalyzed for isoprene formation. Strains were cultivated in duplicate onsolid media (tryptic soy broth agar, TSB; 2.5 ml) in presence or absenceof thiostrepton (200 μg/ml) and incubated for 4 days at 28° C. in sealedhead-space vials (total volume 20 ml). 500 μl head-space samples (endpoint measurements) were analyzed by GC-MS in SIM-mode and isoprene wasidentified according to reference retention times and molecular masses(67 m/z). Isoprene present in head-space samples was quantified bypreviously generated calibration curves. While wild-type S. albus andcontrol strains harboring pUWL201PW produced isoprene in concentrationsslightly higher than the detection limit (0.04-0.07 ppm), S. albusharboring pUWL201_iso produced isoprene in at least tenfold excesscompared to controls (0.75 ppm; FIG. 72). The results demonstratesuccessful expression of plant-derived isoprene synthase in aprokaryotic organism of the Actinomycetes group.

Example 12 Production of Isoprene or Mevalonate from Fatty Acid or PalmOil in E. coli fadR atoC LS5218 Containing the Upper or Upper and LowerMevalonic Acid Pathway Plus Kudzu Isoprene Synthase

Escherichia coli fadR atoC strain LS5218 (#6966) was obtained from theColi Genetic Stock Center. FadR encodes a transcription repressor thatnegatively regulates expression of the genes encoding fatty aciddegradation enzymes (Campbell et al., J. Bacteriol. 183: 5982-5990,2001). AtoC is a response regulator in a two-component regulatory systemwherein AtoS regulates acetolactate metabolism. The fadR atoC strainallows constitutive expression of the fatty acid degradation genes andincorporates long chain fatty acids into long-chain-lengthpolyhydroxyalkanoates. When palm oil is used as a carbon source foreither mevalonate or isoprene production, the palm oil was converted toglycerol plus fatty acid. Methods for this are well known in the art,and it can be done either enzymatically by incubation with a lipase (forexample Porcine pancreatic lipase, Candida rugosa lipase, or othersimilar lipases) or chemically by saponification with a base such assodium hydroxide.

i) E. coli fadR atoC Strain Expressing the Upper Mevalonic Acid Pathway

Strain WW4 was created by electroporating pCLPtrcUpperPathway intoLS5218 using standard methods (Sambrooke et al., Molecular Cloning: ALaboratory Manual, 2^(nd) ed., Cold Spring Harbor, 1989). Incorporationof the plasmid was demonstrated by the production of mevalonic acid(MVA) when cells were cultured in TM3 medium supplemented with eitherC12 fatty acid (FA) or palm oil as the carbon source. To demonstrateproduction of MVA by WW4 from fatty acid, cells from an overnightculture were diluted 1 to 100 into 5 mL of modified TM3 medium (TM3without yeast extract) supplemented with 0.25% C12 FA (Sigma cat#L9755). The first sign of MVA production (24 mg/L) was apparent afterovernight incubation at 30° C. of the IPTG induced culture. Productionincreased over three days with the final level of 194 mg/L of MVAproduced. To demonstrate production of MVA by WW4 from oil, cells froman overnight culture were diluted 1 to 100 into modified TM3 mediumsupplemented with 200 mg of digested palm oil per 5 mL of TM3 medium.The first sign of MVA production (50 mg/L) was apparent after overnightincubation of the IPTG induced culture at 30° C. Production increasedover three days with a final level of 500 mg/L of MVA produced.

ii) E. coli fadR atoC Strain Expressing the Upper and Lower MVA PathwayPlus Kudzu Isoprene Synthase

Escherichia coli strain WW4 (LS5218 fadR atoC pCLPtrcUpperPathway) wastransformed with pMCM118 [pTrcKKDyIkIS] to yield WW10. The incorporationof the plasmid was demonstrated by evidence of production of isoprenewhen the strain was cultured in TM3 and glucose and induced with IPTG(100, 300, or 900 uM). The strain was relatively sensitive to IPTG andshowed a significant growth defect even at 100 uM IPTG. These resultsare shown in FIG. 70A.

To test isoprene production from dodecanoic acid, WW10 was culturedovernight in L broth containing spectinomycin (50 ug/ml), and kanamycin(50 ug/ml) at 37 C with shaking at 200 rpm. The cells were washed withmodified TM3 medium by centrifugation and resuspension in their originalculture volume with this medium. The washed and resuspended cells fromthis starter culture were diluted 1 to 100 and 1 to 10 into 5 mL ofmodified TM3 medium containing 0.125% C12 Fatty Acid (Sigma cat #L9755).

To demonstrate production of mevalonate from palm oil, the oil waspredigested with lipase at 37° C. and 250 rpm for several days torelease the fatty acids (evidence of hydrolysis was judged by the foamformed when tubes were shaken).

In addition, a culture was set up by diluting the washed cells at 1 to10 into modified TM3 medium contained in test tubes with palm oil. Afurther tube was set up by the addition of 0.125% C12FA to the remainder(2.5 mL) of the washed cells without further dilution (bioconversion).After 3.75 hours of growth at 30° C. with shaking at 250 rpm all of thecultures were induced by the addition of 50 uM IPTG. Incubation wascontinued for 4 hours after which time 200 uL of each of the cultureswas assayed for isoprene accumulation with a modified head space assay(1 hour accumulation at 30° C. with shaking at 500 rpm). An additionalisoprene assay was conducted by a 12 hour incubation of the assay glassblock prior to GCMS analysis. Incubation of the induced cultures wascontinued overnight and 200 uL aliquots were again assayed for isopreneproduction (1 hour, 30 deg, 500 rpm Shel-shaker) the following morning.Analysis of these cultures showed the production of significant levelsof isoprene. The highest levels of isoprene were observed in the culturewhich was seeded at 1/10 dilution from the overnight starter cultureafter it had been incubated and induced overnight. This result suggeststhat this culture continued to grow and increase in cell density. Theseresults are shown in FIG. 70B. Cell density could not be measureddirectly because the fatty acid suspension had a turbid appearance. Celldensity of this culture was therefore determined by plating an aliquotof the culture and showed 8×10⁷ colony forming units. This correspondsapproximately to an OD₆₀₀ of 0.1. Nevertheless, this culture providedsignificant isoprene production; no isoprene is observed for similarstrains without the pathway described in this example.

Example 13 Improvement of Isoprene Production by Constitutive Expressionof ybhE in E. coli.

This example shows production of isoprene in a strain constitutivelyexpressing ybhE (pgl) compared to a control strain with wild type ybhE.The gene ybhE (pgl) encodes a 6-phosphogluconolactonase that suppressesposttranslational gluconylation of heterologously expressed proteins andimproves product solubility and yield while also improving biomass yieldand flux through the pentose phosphate pathway (Aon et al. Applied andEnvironmental Microbiology, 74(4): 950-958, 2008).

The BL21 strain of E. coli producing isoprene (EWL256) was constructedwith constitutive expression of the ybhE gene on a replicating plasmidpBBR1MCS5(Gentamycin) (obtained from Dr. K. Peterson, Louisiana StateUniversity).

FRT-based recombination cassettes, and plasmids for Red/ET-mediatedintegration and antibiotic marker loopout were obtained from GeneBridges GmbH (Germany). Procedures using these materials were carriedout according to Gene Bridges protocols. Primers Pgl-F and PglGI1.5-Rwere used to amplify the resistance cassette from the FRT-gb2-Cm-FRTtemplate using Stratagene Herculase II Fusion kit according to themanufacturer's protocol. The PCR reaction (50 uL final volume)contained: 5 uL buffer, 1 uL template DNA (FRT-gb2-Cm-F from GeneBridges), 10 pmols of each primer, and 1.5 uL 25 mM dNTP mix, made to 50uL with dH₂O. The reaction was cycled as follows: 1×2 minutes, 95° C.then 30 cycles of (30 seconds at 95° C.; 30 seconds at 63° C.; 3 minutesat 72° C.).

The resulting PCR product was purified using the QiaQick PCRpurification kit (Qiagen) and electroporated into electrocompetentMG1655 cells harboring the pRed-ET recombinase-containing plasmid asfollows. Cells were prepared by growing in 5 mLs of L broth to andOD600-0.6 at 30° C. The cells were induced for recombinase expression bythe addition of 4% arabinose and allowed to grow for 30 minutes at 30°C. followed by 30 minutes of growth at 37° C. An aliquot of 1.5 mLs ofthe cells was washed 3-4 times in ice cold dH₂O. The final cell pelletwas resuspended in 40 uL of ice cold dH₂O and 2-5 uL of the PCR productwas added. The electroporation was carried out in 1-mm gap cuvettes, at1.3 kV in a Gene Pulser Electroporator (Bio-Rad Inc.). Cells wererecovered for 1-2 hours at 30° C. and plated on L agar containingchloramphenicol (5 ug/mL). Five transformants were analyzed by PCR andsequencing using primers flanking the integration site (2 primer sets:pgl and 49 rev and 3′ EcoRV-pglstop; Bottom Pgb2 and Top GB's CMP(946)). A correct transformant was selected and this strain wasdesignated MG1655 GI1.5-pgl::CMP.

The chromosomal DNA of MG1655 GI1.5-pgl::CMP was used as template togenerate a PCR fragment containing the FRT-CMP-FRT-GI1.5—ybhE construct.This construct was cloned into pBBR1MCS5(Gentamycin) as follows. Thefragment, here on referred to as CMP-GI1.5-pgl, was amplified using the5′ primer Pglconfirm-F and 3′ primer 3′ EcoRV-pglstop. The resultingfragment was cloned using the Invitrogen TOPO-Blunt cloning kit into theplasmid vector pCR-Blunt II-TOPO as suggested from the manufacturer. TheNsiI fragment harboring the CMP-GI1.5-pgl fragment was cloned into thePstI site of pBBR1MCS5(Gentamycin). A 20 μl ligation reaction wasprepared containing 5 μl CMP-GI1.5-pgl insert, 2 μlpBBR1MCS5(Gentamycin) vector, 1 μl T4 DNA ligase (New England Biolabs),2 μl 10× ligase buffer, and 10 μl ddH₂O. The ligation mixture wasincubated at room temperature for 40 minutes then 2-4 uL wereelectroporated into electrocompetent Top10 cells (Invitrogen) using theparameters disclosed above. Transformants were selected on L agarcontaining 10 ug/ml chloramphenicol and 5 ug/ml Gentamycin. The sequenceof the selected clone was determined using a number of the primersdescribed above as well as with the in-house T3 and Reverse primersprovided by Sequetech, Calif. This plasmid was designatedpBBRCMPGI1.5-pgl (FIGS. 77A-B and SEQ ID NO:122).

Plasmid pBBRCMPGI1.5-pgl was electroporated into EWL256, as describedabove in Example 10 and transformants were plated on L agar containingChloramphenicol (10 ug/mL), Gentamycin (5 ug/mL), spectinomycin (50ug/mL), and carbenicillin (50 ug/mL). One transformant was selected anddesignated RM11608-2.

Primers: Pgl-F (SEQ ID NO: 115)5′-ACCGCCAAAAGCGACTAATTTTAGCTGTTACAGTCAGTTGAATTAACCCTCACTAAAGGGCGGCCGC-3′ PglGI1.5-R (SEQ ID NO: 116)5′-GCTGGCGATATAAACTGTTTGCTTCATGAATGCTCCTTTGGGTTACCTCCGGGAAACGCGGTTGATTTGTTTAGTGGTTGAATTATTTGCTCAGGATGTGGCATAGTCAAGGGCGTGACGGCTCGCTAATACGACTCACTA TAGGGCTCGAG-3′ 3′EcoRV-pglstop: (SEQ ID NO: 117)5′-CTT GAT ATC TTA GTG TGC GTT AAC CAC CAC pgl +49rev: (SEQ ID NO: 118)CGTGAATTTGCTGGCTCTCAG Bottom Pgb2: (SEQ ID NO: 119)GGTTTAGTTCCTCACCTTGTC Top GB's CMP (946): (SEQ ID NO: 120)ACTGAAACGTTTTCATCGCTC Pglconfirm-F (SEQ ID NO: 121)5′-ACCGCCAAAAGCGACTAATTTTAGCT-3′i) Small Scale AnalysisMedia Recipe (Per Liter Fermentation Media):

K₂HPO₄ 13.6 g, KH₂PO₄ 13.6 g, MgSO₄*7H₂O 2 g, citric acid monohydrate 2g, ferric ammonium citrate 0.3 g, (NH₄)₂SO₄ 3.2 g, yeast extract 1 g,1000× Trace Metals Solution 1 ml. All of the components were addedtogether and dissolved in diH₂O. The pH was adjusted to 6.8 withammonium hydroxide (30%) and brought to volume. Media wasfilter-sterilized with a 0.22 micron filter. Glucose 5.0 g andantibiotics were added after sterilization and pH adjustment.

1000× Trace Metal Solution (Per Liter Fermentation Media):

Citric Acid*H₂O 40 g, MnSO₄*H₂O 30 g, NaCl 10 g, FeSO₄*7H₂O 1 g,CoCl₂*6H₂O 1 g, ZnSO₄*7H₂O 1 g, CuSO₄*5H₂O 100 mg, H₃BO₃ 100 mg,NaMoO₄*2H₂O 100 mg. Each component is dissolved one at a time in diH₂O.The pH is adjusted to 3.0 with HCl/NaOH, and then the solution isbrought to volume and filter-sterilized with a 0.22 micron filter.

a) Experimental Procedure

Isoprene production was analyzed by growing the strains in a Cellerator™from MicroReactor Technologies, Inc. The working volume in each of the24 wells was 4.5 mL. The temperature was maintained at 30° C., the pHsetpoint was 7.0, the oxygen flow setpoint was 20 sccm and the agitationrate was 800 rpm. An inoculum of E. coli strain taken from a frozen vialwas streaked onto an LB broth agar plate (with antibiotics) andincubated at 30° C. A single colony was inoculated into media withantibiotics and grown overnight. The bacteria were diluted into 4.5 mLof media with antibiotics to reach an optical density of 0.05 measuredat 550 nm.

Off-gas analysis of isoprene was performed using a gaschromatograph-mass spectrometer (GC-MS) (Agilent) headspace assay.Sample preparation was as follows: 100 μL of whole broth was placed in asealed GC vial and incubated at 30° C. for a fixed time of 30 minutes.Following a heat kill step, consisting of incubation at 70° C. for 5minutes, the sample was loaded on the GC.

Optical density (OD) at a wavelength of 550 nm was obtained using amicroplate reader (Spectramax) during the course of the run. Specificproductivity was obtained by dividing the isoprene concentration (μg/L)by the OD reading and the time (hour).

The two strains EWL256 and RM11608-2 were assessed at 200 and 400 uMIPTG induction levels. Samples were analyzed for isoprene production andcell growth (OD550) at 1, 2.5, 4.75, and 8 hours post-induction. Sampleswere done in duplicate.

b) Results

The experiment demonstrated that at 2 different concentrations of IPTGthe strain expressing the ybhE (pgl) had a dramatic 2-3 fold increase inspecific productivity of isoprene compared to the control strain.

ii) Isoprene Fermentation from E. coli Expressing M. mazei MevalonateKinase, P. alba Isoprene Synthase, and pgl Over-Expression (RHM111608-2)and Grown in Fed-Batch Culture at the 15-L Scale

Medium Recipe (Per Liter Fermentation Medium)

K₂HPO₄ 7.5 g, MgSO₄*7H₂O 2 g, citric acid monohydrate 2 g, ferricammonium citrate 0.3 g, yeast extract 0.5 g, 1000× Modified Trace MetalSolution 1 ml. All of the components were added together and dissolvedin diH₂O. This solution was autoclaved. The pH was adjusted to 7.0 withammonium hydroxide (30%) and q.s. to volume. Glucose 10 g, thiamine*HCl0.1 g, and antibiotics were added after sterilization and pH adjustment.

1000× Modified Trace Metal Solution:

Citric Acids*H₂O 40 g, MnSO₄*H₂O 30 g, NaCl 10 g, FeSO₄*7H₂O 1 g,CoCl₂*6H₂O 1 g, ZnSO₄*7H₂O 1 g, CuSO₄*5H₂O 100 mg, H₃BO₃ 100 mg,NaMoO₄*2H₂O 100 mg. Each component is dissolved one at a time in Di H₂O,pH to 3.0 with HCl/NaOH, then q.s. to volume and filter sterilized witha 0.22 micron filter.

Fermentation was performed in a 15-L bioreactor with BL21 (DE3) E. colicells containing the upper mevalonic acid (MVA) pathway (pCL Upper), theintegrated lower MVA pathway (gi1.2KKDyI), high expression of mevalonatekinase from M. mazei and isoprene synthase from P. alba (pTrcAlba-mMVK),and high expression of pgl (pBBR-pgl). This experiment was carried outto monitor isoprene formation from glucose at the desired fermentationpH 7.0 and temperature 34° C. A frozen vial of the E. coli strain wasthawed and inoculated into tryptone-yeast extract medium. After theinoculum grew to OD 1.0, measured at 550 nm, 500 mL was used toinoculate a 15-L bioreactor bringing the initial volume to 5-L.

Glucose was fed at an exponential rate until cells reached thestationary phase. After this time the glucose feed was decreased to meetmetabolic demands. The total amount of glucose delivered to thebioreactor during the 40 hour (59 hour) fermentation was 3.1 kg (4.2 kgat 59 hour). Induction was achieved by adding IPTG. The IPTGconcentration was brought to 110 uM when the optical density at 550 nm(OD₅₅₀) reached a value of 4. The IPTG concentration was raised to 192uM when OD₅₅₀ reached 150. The OD₅₅₀ profile within the bioreactor overtime is shown in FIG. 78A. The isoprene level in the off gas from thebioreactor was determined using a Hiden mass spectrometer. The isoprenetiter increased over the course of the fermentation to a maximum valueof 33.2 g/L at 40 hours (48.6 g/L at 59 hours) (FIG. 78B). The isoprenetiter increased over the course of the fermentation to a maximum valueof 40.0 g/L at 40 hours (60.5 g/L at 59 hours) (FIG. 78C). The totalamount of isoprene produced during the 40-hour (59-hour) fermentationwas 281.3 g (451.0 g at 59 hours) and the time course of production isshown in FIG. 78D. The time course of volumetric productivity is shownin FIG. 78E and shows that an average rate of 1.0 g/L/hr was maintainedbetween 0 and 40 hours (1.4 g/L/hour between 19 and 59 hour). Themetabolic activity profile, as measured by CER, is shown in FIG. 78F.The molar yield of utilized carbon that went into producing isopreneduring fermentation was 19.6% at 40 hours (23.6% at 59 hours). Theweight percent yield of isoprene from glucose was 8.9% at 40 hours(10.7% at 59 hours).

Preparation of Isoprene Samples for Polymerization

(a) Preparation of 1000× Modified Trace Metal Solution:

Each of the following components is dissolved one at a time in Di H₂O:Citric Acid*H₂O (40 g), MnSO₄*H₂O (30 g), NaCl (10 g), FeSO₄*7H₂O (1 g),CoCl₂*6H₂O (1 g), ZnSO*7H₂O (1 g), CuSO₄*5H₂O (100 mg), H₃BO₃ (100 mg),NaMoO₄*2H₂O (100 mg). The pH was adjusted to 3.0 with HCl/NaOH, thenq.s. to volume and filter sterilized with a 0.22 micron filter.

(b) Preparation of Fermentation Medium:

Each liter of fermentation medium contained K₂HPO₄ (7.5 g), MgSO₄*7H₂O(2 g), citric acid monohydrate (2 g), ferric ammonium citrate (0.3 g),yeast extract (0.5 g), 1000× Modified Trace Metal Solution (1 ml). Allof the components were added together and dissolved in diH2O. Thissolution was autoclaved. The pH was adjusted to 7.0 with ammonium gas(NH3) and q.s. to volume. Glucose (10 g), thiamine*HCl (0.1 g), andantibiotic were added after sterilization and pH adjustment.

(c) Collection of Isoprene Samples for Purification and Polymerization:

Isoprene was collected by adsorption on activated charcoal by passingthe fermentation exhaust across canisters of activated charcoal arrangedin parallel on an exhaust manifold.

(d) Preparation of Isoprene Polymerization Sample A from Glucose usingE. coli

Fermentation was performed at pH 7.0 and 30° C. in a 15-L bioreactorwith BL21 (DE3) E. coli cells containing the pCL PtrcUpperMVA and pTrcKKDyIkIS plasmids. An inoculum of E. coli strain taken from a frozenvial was streaked onto an LB broth agar plate (with antibiotics) andincubated at 37° C. A single colony was inoculated into tryptone-yeastextract medium. After the inoculum grew to OD 1.0, measured at 550 nm,500 mL was used to inoculate a 5-L bioreactor.

Glucose was fed at an exponential rate until cells reached thestationary phase. After this time the glucose feed was decreased to meetmetabolic demands. The total amount of glucose delivered to thebioreactor during the 54 hour fermentation was 3.7 kg. Induction wasachieved by adding isopropyl-beta-D-1-thiogalactopyranoside (IPTG). TheIPTG concentration was brought to 25 μM when the optical density at 550nm (OD₅₅₀) reached a value of 10. The IPTG concentration was raised to50 uM when OD550 reached 190. IPTG concentration was raised to 100 uM at38 hours of fermentation. The OD550 profile within the bioreactor overtime is shown in FIG. 1. The isoprene titer increased over the course ofthe fermentation to a final value of 2.2 g/L (FIG. 2). The total amountof isoprene produced during the 54 hour fermentation was 15.9 g and thetime course of production is shown in FIG. 3. The molar yield ofutilized carbon that went into producing isoprene during fermentationwas 1.53%. (See FIGS. 80, 81 and 82).

(e) Preparation of Isoprene Polymerization Sample B from Glucose andYeast Extract Using E. coli

Isoprene formation from glucose and yeast extract was performed at pH7.0 and 30° C. in a 500-L bioreactor with E. coli cells containing thepTrcKudzu+yIDI+DXS plasmid. An inoculum of E. coli strain taken from afrozen vial was prepared in soytone-yeast extract-glucose medium. Afterthe inoculum grew to OD 0.15, measured at 550 nm, 20 mL was used toinoculate a bioreactor containing 2.5-L tryptone-yeast extract medium.The 2.5-L bioreactor was grown at 30° C. to OD 1.0 and 2.0-L wastransferred to the 500-L bioreactor. Yeast extract (Bio Springer,Montreal, Quebec, Canada) and glucose were fed at exponential rates. Thetotal amount of glucose and yeast extract delivered to the bioreactorduring the 50 hour fermentation was 181.2 kg and 17.6 kg, respectively.The optical density within the bioreactor over time is shown in FIG. 4.The isoprene titer increased over the course of the fermentation (FIG.5). The total amount of isoprene produced during the 50 hourfermentation was 55.1 g and the time course of production is shown inFIG. 6.

Isoprene Desorption from Activated Charcoal (Method A)

Activated charcoal (130 g), which had been exposed to a stream offermentor off-gas, was placed into a 1000 mL flask along with a stirbar. Cyclohexane (563 mL) was added to the flask and the slurry wasagitated for 2 hours. Vacuum was applied (100 mbar) via an in-linecryogenic trap (30 mL capacity, immersed in liq. nitrogen). Fourfractions were collected and combined to yield an isoprene/cyclohexanesolution (2.1 wt % isoprene, total volume=53.1 g). This solution wasvacuum distilled at 100 mbar and a new isoprene/cyclohexane solution wascollected (yield=10.1 g), which was dried over 3 A molecular sieves. GCanalysis of this solution indicated an isoprene content of 7.7 wt. %.

Isoprene Desorption from Activated Charcoal (Method B)

Activated charcoal (65 g), which had been exposed to a stream offermentor off-gas, was placed into a 500 mL flask along with a stir bar.Jarytherm DBT (250 g) was added to the charcoal and the slurry wasagited for 2 hours. Vacuum was applied (5 mbar) via an in-line cryogenictrap (30 mL capacity, immersed in liq. nitrogen). After 1 hour the trapwas warmed to ambient temperature. Two liquid phases were present in thetrap (total weight 1.82 g). The organic phase was diluted withcyclohexane (3.26 g), decanted, and dried over 3 A molecular sieves. GCanalysis of this solution indicated an isoprene content of 27.3 wt. %,or 1.22 g).

Preparation of Neodymium Catalyst

Neodymium versatate (2.68 mL, 0.51 M in hexane,), triisobutylaluminum(54 mL, 1.0 M in hexane), and diethylaluminum chloride (3.40 mL, 1.0 Min hexane) were drawn up into plastic syringes fitted with steelcannula. The first two components were added to a solution of1,3-butadiene in hexane (22.4 mL, 15 wt. % 1,3-butadiene, placed into a100 mL glass vessel with septum top, and agitated for 0.5 h at ambienttemperature. The last component was added to the solution after which itwas heat-aged for 0.5 h at 65° C. The final solution was clear andyellow. The concentration of the solution based on neodymium was 0.0164M.

Preparation of Titanium Catalyst

A 100 mL glass reaction vessel with septum inlet and containing amagnetic stirbar was placed in an ice bath at 0° C., charged withn-hexane (5.07 mL, anhydrous), and with neat TiCl₄ (1.5 mL, 13.7 mmol)under vigorous agitation. Separately, a solution was generatedconsisting of diphenyl ether (1.2 mL, 7.6 mmol) and triisobutylaluminum(14.6 mL, 12.6 mmol, 25 wt. % solution in hexane). The solution wasadded to the reaction vessel over the course of 5 minutes. A brownprecipitate formed during the addition. The suspension was stirred for10 minutes and was then stored at −40° C. for future use.

Polymerization

Samples of polyisoprene derived primarily from glucose were produced bypolymerizing Isoprene Polymerization Sample A with Neodymium catalystand n-BuLi. Samples of polyisoprene derived from cofermentation ofglucose and yeast extract were produced by polymerizing IsoprenePolymerization Sample B with Neodymium catalyst, titatium catalyst,n-BuLi catalyst, and emulsion free radical polymerization.Representative polymerization conditions are described below.

Solution Polymerization of Isoprene with Neodymium Catalyst

A 4 mL screw top glass vial with Teflon coated stir bar was annealed inan oven for 3 h at 150° C. The vial was fitted with a Teflon facedsilicone septum and open-top cap. Using a syringe, it was then chargedwith an isoprene solution (1.5 g, 7.7 wt. % in cyclohexane, anhydrous).Neodymium catalyst solution (60 uL) was injected into the vial with amicro-syringe. The vial was placed onto a stirrer/hotplate regulated to65° C., with the stir bar spinning at 500 rpm. After 15 minutes thesolution became noticeably more viscose. After a reaction time of 1.5 hthe reaction was quenched with a solution of isopropanol and butylatedhydroxytoluene, (BHT), (30 uL, 10 wt. % BHT). A 100 mg sample of thecement was removed for GPC analysis. The remaining polymer cement wasdried under ambient conditions. The isolated polymer weighed 110 mg, wasdetermined to have a weight average molecular weight of 935,000 (by GPC)and a cis-mirrostructure content of greater than 90% (by ¹³C-NMR).

Solution Polymerization of Isoprene with Ti Catalyst

A 4 mL screw top glass vial and Teflon coated stir bar was annealed inan oven for 3 h at 150° C. The vial was fitted with a pre-scored Teflonfaced silicone septum and open-top cap. Using a syringe, it was thencharged with an isoprene solution (1.5 g, 7.7 wt. % in cyclohexane,anhydrous). The titanium catalyst suspension was magnetically stirredand a sample was removed (70 uL) with a disposable tip pipette, whichwas then added to the reaction vial through the pre-scored septum. Thereaction vial septum was replaced with a solid cap, and the vial wasplaced onto a stirrer/hotplate'regulated to 65° C., with the stir barspinning at 500 rpm. After 5 minutes the solution became noticeably moreviscose. After a reaction time of 1.5 h the reaction was quenched with asolution of isopropanol and butylated hydroxytoluene, (BHT), (30 uL. 10wt. % BHT). A 100 mg sample of the cement was removed for GPC analysis.The remaining polymer cement was dried under ambient conditions. Theisolated polymer weighed 108 mg, had a weight average molecular weightof 221,000 (by GPC), and had a cis-mirocstructure content of greaterthan 94% (by ¹³C-NMR).

Emulsion Polymerization of Isoprene

A 20 mL vial was used as a polymerization vessel. The metal cap waspierced twice with an awl and cardboard linear was replaced with arubber gasket and Teflon linear. The vial was rinsed with deionied waterand dried under nitrogen.

To the vial was added 7.05 g deionized water, 1.14 g of 10% soap(potassium salt of mixed fatty acids), 174 mg of 10% ammonium persulfatesolution, and 24 mg of n-dodecane thiol. The flask was purge for 30seconds with nitrogen and capped. To the vial through the rubber/Teflongasket was charged 3 mL of bio-HG (2.033 grams of isoprene). The vialwas placed in a standard bottle polymerization bath (a second blank vialallows the vial to fit in a 4 oz bottle holder). The mixture was tumbledfor 25.5 hours at 65° C. (+/−0.2° C.).

Work-Up:

The latex was transferred to 50 mL pear shaped flask and diluted with 10mL of water. Un-reacted volatile organic was removed by evaporating 2 mLof water under vacuum (54 mmHg, 40-50° C.). To the latex was added anantioxidant dispersion, 140 mg of 50% active polyphenolic AO (Bostex24). The latex was coagulated by adding it to a dilute acid solution (12mL of 18% sulfuric acid in 150 mL RO water). The polymer coagulated intoa single large piece which was pressed and washed with RO water. Thesample was off white soft rubbery mass. The yield was 1.24 grams of wetcrumb.

The final total solids content (TSC=100*dried weight/wet weight) was18.9 wt % or an approximate conversion of 84%.

Polymerization of Isoprene with Butyllithium

Butyllithium (1.6 M in hexane) was diluted with n-hexane (anhydrous) ina ratio of 1:10. The solution was titrated against a standardN-pivalolyl-o-benzylaniline in THF. A solution of isoprene incyclohexane (4 mL) was dried by passing it through a small columncontaining heat treated silica gel.

A 4 mL glass vial (oven dried at 150° C.) was charged with a smallTeflon coated magnetic stir bar and a solution of isoprene incyclohexane (1.35 g, 21.5 wt %). Butyllithium (0.14 M, hexane) was addedvia syringe and the vial was heated to 65° C. on a stirrer/hot plate for3 h. The polymer reaction was quenched with a BHT/iso-propanol solution(10 wt % BHT in iso-propanol). All volatiles were removed under vacuum.This procedure yielded 290 mg of polymer which reprecsents a theoreticalyield of about 100%. This polymer was determined by GPC analysis to havea weight average molecular weight (M_(w)) of 17,880 and was determinedby ¹³C NMR to have a cis-microstructure content of 67%; atrans-microstructure content of 25%, and a 3,4-microstructure content of8%.

GPC Analysis of Polymers

Size Exclusion Chromatography (SEC) is a well established technique tomeasure polymer molecular weight and polydispersity (Mw/Mn). Two PolymerLaboratories C microgel columns in series were utilized withtetrahydrofuran as the carrier solvent at a flow rate of 0.7 ml/min anda column temperature of 40° C. SEC was performed using a WyattTechnologies miniDawn light scattering detector coupled with a HewlettPackard 1047A refractive index detector. Polystyrene standards were usedto calibrate the instrument.

NMR Analysis of Polymers

Polymer microstructures were determined by ¹³C-NMR analysis on a VarianUnity-Plus 400 MHz spectrometer in chloroform-di solvent.

Data from ¹³C/¹²C Isotope Analyses

Entry Sample δ¹³C 1 PI from sugar beet invert sugar −34.98 2 CommercialPI from isobutylene −34.43 3 Commercial PI from isobutylene −34.42 4Guayule rubber −31.10 5 Palm oil −30.03 6 Palm oil −30.00 7 Naturalrubber (Neco) −28.11 8 Natural rubber (Pumpic) −27.92 9 Natural rubber(Negato) −27.86 10 Natural rubber (Nivco) −27.79 11 Natural rubber(Naplo) −27.74 12 Natural rubber (Krado 1) −27.68 13 Natural rubber(Krado 1) −27.55 14 Natural rubber (Krado 2) −27.54 15 Natural rubber(Krado 2) −27.52 16 Natural rubber (Krado 2) −27.49 17 Natural rubber(Nolo) −27.38 18 Yeast extract −25.70 19 Yeast extract −25.68 20Commercial PI from extractive distillation (Sample 2) −23.83 21Commercial PI from extractive distillation (Sample 2) −23.83 22 Sugarfrom softwood pulp (Sample 2) −23.25 23 Sugar from softwood pulp(Sample 1) −23.00 24 Sugar from softwood pulp (Sample 1) −22.96 25Commercial PI from extractive distillation (Sample 3) −22.95 26Commercial PI from extractive distillation (Sample 3) −22.95 27Commercial PI from extractive distillation (Sample 3) −22.94 28Commercial PI from extractive distillation (Sample 3) −22.92 29Commercial PI from extractive distillation (Sample 3) −22.90 30Commercial PI from extractive distillation (Sample 3) −22.89 31Commercial PI from extractive distillation (Sample 3) −22.89 32Commercial PI from extractive distillation (Sample 3) −22.89 33Commercial PI from extractive distillation (Sample 3) −22.87 34Commercial PI from extractive distillation (Sample 3) −22.84 35Commercial PI from extractive distillation (Sample 1) −22.63 36Commercial PI from extractive distillation (Sample 1) −22.62 37Commercial PI from extractive distillation (Sample 1) −22.54 38 PI fromIsoprene Sample B (emulsion polymerization) −19.67 39 PI from IsopreneSample B (Neodymium catalyst) −19.14 40 PI from Isoprene Sample B(Neodymium catalyst) −18.80 41 PI from Isoprene Sample B (Neodymiumcatalyst) −18.37 42 PI from Isoprene Sample B (n-BuLi catalyst) −18.1243 PI from Isoprene Sample B (n-BuLi catalyst) −18.12 44 Invert Sugar(Sample 1) −15.37 45 Invert Sugar (Sample 2) −15.36 46 Invert Sugar(Sample 1) −15.34 47 Invert Sugar (Sample 1) −15.31 48 Invert Sugar(Sample 1) −15.25 49 PI from Isoprene Sample A (Neodymium catalyst)−14.85 50 PI from Isoprene Sample A (n-BuLi catalyst) −14.69 51 PI fromIsoprene Sample A (n-BuLi catalyst) −14.69 52 PI from Isoprene Sample A(n-BuLi catalyst) −14.66 53 Glucose from bagasse (sample 2) −13.19 54Glucose from bagasse (sample 1) −13.00 55 Glucose from bagasse(sample 1) −12.93 56 Glucose from corn stover (sample 2) −11.42 57Glucose from corn stover (sample 1) −11.23 58 Glucose from corn stover(sample 1) −11.20 59 Cornstarch −11.12 60 Cornstarch −11.11 61Cornstarch −11.10 62 Cornstarch −11.07 63 Glucose −10.73 (note: PI =polyisoprene)

Unless defined otherwise, the meanings of all technical and scientificterms used herein are those commonly understood by one of skill in theart to which this invention pertains. Singleton, et al., Dictionary ofMicrobiology and Molecular Biology, 2nd ed., John Wiley and Sons, NewYork (1994), and Hale & Marham, The Harper Collins Dictionary ofBiology, Harper Perennial, N.Y. (1991) provide general definitions formany of the terms used herein.

The headings provided herein are not limitations of the various aspectsor embodiments of the invention which can be had by reference to thespecification as a whole. For use herein, unless clearly indicatedotherwise, use of the terms “a”, “an,” and the like refers to one ormore. Reference to “about” a value or parameter herein includes (anddescribes) embodiments that are directed to that value or parameter perse. For example, description referring to “about X” includes descriptionof “X.” Numeric ranges are inclusive of the numbers defining the range.It is understood that aspects and embodiments of the invention describedherein include “comprising,” “consisting,” and “consisting essentiallyof” aspects and embodiments.

While certain representative embodiments and details have been shown forthe purpose of illustrating the subject invention, it will be apparentto those skilled in this art that various changes and modifications canbe made therein without departing from the scope of the subjectinvention. It is to be understood that this invention is not limited tothe particular methodology, protocols, and reagents described, as thesemay vary. One of skill in the art will also appreciate that any methodsand materials similar or equivalent to those described herein can alsobe used to practice or test the invention.

Appendix 1 Exemplary 1-deoxy-D-xylulose-5-phosphate Synthase NucleicAcids and Polypeptides

-   ATH: AT3G21500(DXPS1) AT4G15560(CLA1) AT5G11380(DXPS3)-   OSA: 4338768 4340090 4342614-   CME: CMF089C-   PFA: MAL13P1.186-   TAN: TA20470-   TPV: TP01_(—)0516-   ECO: b0420(dxs)-   ECJ: JWO410(dxs)-   ECE: Z0523(dxs)-   ECS: ECs0474-   ECC: c0531(dxs)-   ECI: UTI89_C0443(dxs)-   ECP: ECP_(—)0479-   ECV: APECO1_(—)1590(dxs)-   ECW: EcE24377A_(—)0451(dxs)-   ECX: EcHS_A0491-   STY: STY0461(dxs)-   STT: t2441(dxs)-   SPT: SPA2301(dxs)-   SEC: SC0463(dxs)-   STM: STM0422(dxs)-   YPE: YPO3177(dxs)-   YPK: y1008(dxs)-   YPM: YP_(—)0754(dxs)-   YPA: YPA_(—)2671-   YPN: YPN_(—)0911-   YPP: YPDSF_(—)2812-   YPS: YPTB0939(dxs)-   YPI: YpsIP31758_(—)3112(dxs)-   SFL: SF0357(dxs)-   SFX: S0365(dxs)-   SFV: SFV_(—)0385(dxs)-   SSN: SSON_(—)0397(dxs)-   SBO: SBO_(—)0314(dxs)-   SDY: SDY_(—)0310(dxs)-   ECA: ECA1131(dxs)-   PLU: plu3887(dxs)-   BUC: BU464(dxs)-   BAS: BUsg448(dxs)-   WBR: WGLp144(dxs)-   SGL: SG0656-   KPN: KPN_(—)00372(dxs)-   BFL: Bfl238(dxs)-   BPN: BPEN_(—)244(dxs)-   HIN: HI1439(dxs)-   HIT: NTHI1691(dxs)-   HIP: CGSHiEE_(—)04795-   HIQ: CGSHiGG_(—)01080-   HDU: HD0441(dxs)-   HSO: HS_(—)0905(dxs)-   PMU: PM0532(dxs)-   MSU: MS1059(dxs)-   APL: APL_(—)0207(dxs)-   XFA: XF2249-   XFT: PD1293(dxs)-   XCC: XCC2434(dxs)-   XCB: XC_(—)1678-   XCV: XCV2764(dxs)-   XAC: XAC2565(dxs)-   XOO: XOO2017(dxs)-   XOM: XOO_(—)1900(XOO1900)-   VCH: VC0889-   VVU: VV1_(—)0315-   VVY: VV0868-   VPA: VP0686-   VFI: VF0711-   PPR: PBPRA0805-   PAE: PA4044(dxs)-   PAU: PA14_(—)11550(dxs)-   PAP: PSPA7_(—)1057(dxs)-   PPU: PP_(—)0527(dxs)-   PST: PSPTO_(—)0698(dxs)-   PSB: Psyr_(—)0604-   PSP: PSPPH_(—)0599(dxs)-   PFL: PFL_(—)5510(dxs)-   PFO: Pfl_(—)5007-   PEN: PSEEN0600(dxs)-   PMY: Pmen_(—)3844-   PAR: Psyc_(—)0221(dxs)-   PCR: Pcryo_(—)0245-   ACI: ACIAD3247(dxs)-   SON: SO_(—)1525(dxs)-   SDN: Sden_(—)2571-   SFR: Sfri_(—)2790-   SAZ: Sama_(—)2436-   SBL: Sbal_(—)1357-   SLO: Shew_(—)2771-   SHE: Shewmr4_(—)2731-   SHM: Shewmr7_(—)2804-   SHN: Shewana3_(—)2901-   SHW: Sputw3181_(—)2831-   ILO: IL2138(dxs)-   CPS: CPS_(—)1088(dxs)-   PHA: PSHAa2366(dxs)-   PAT: Patl_(—)1319-   SDE: Sde_(—)3381-   PIN: Ping_(—)2240-   MAQ: Maqu_(—)2438-   MCA: MCA0817(dxs)-   FTU: FTT1018c(dxs)-   FTF: FTF1018c(dxs)-   FTW: FTW_(—)0925(dxs)-   FTL: FTL_(—)1072-   FTH: FTH_(—)1047(dxs)-   FTA: FTA_(—)1131(dxs)-   FTN: FTN_(—)0896(dxs)-   NOC: Noc_(—)1743-   AEH: Mlg_(—)1381-   HCH: HCH_(—)05866(dxs)-   CSA: Csal_(—)0099-   ABO: ABO_(—)2166(dxs)-   AHA: AHA_(—)3321(dxs)-   BCI: BCI_(—)0275(dxs)-   RMA: Rmag_(—)0386-   VOK: COSY_(—)0360(dxs)-   NME: NMB1867-   NMA: NMA0589(dxs)-   NMC: NMC0352(dxs)-   NGO: NGO0036-   CVI: CV_(—)2692(dxs)-   RSO: RSc2221(dxs)-   REU: Reut_A0882-   REH: H16_A2732(dxs)-   RME: Rmet_(—)2615-   BMA: BMAA0330(dxs)-   BMV: BMASAVP1_(—)1512(dxs)-   BML: BMA10299_(—)1706(dxs)-   BMN: BMA10247_A0364(dxs)-   BXE: BxeB2827-   BUR: Bcep18194_B2211-   BCN: Bcen_(—)4486-   BCH: Bcen2424_(—)3879-   BAM: Bamb_(—)3250-   BPS: BPSS1762(dxs)-   BPM: BURPS1710b_A0842(dxs)-   BPL: BURPS1106A_A2392(dxs)-   BPD: BURPS668_A2534(dxs)-   BTE: BTH_II0614(dxs)-   BPE: BP2798(dxs)-   BPA: BPP2464(dxs)-   BBR: BB1912(dxs)-   RFR: Rfer_(—)2875-   POL: Bpro_(—)1747-   PNA: Pnap_(—)1501-   AJS: Ajs_(—)1038-   MPT: Mpe_A2631-   HAR: HEAR0279(dxs)-   MMS: mma_(—)0331-   NEU: NE1161(dxs)-   NET: Neut_(—)1501-   NMU: Nmul_A0236-   EBA: ebA4439(dxs)-   AZO: azo1198(dxs)-   DAR: Daro_(—)3061-   TBD: Tbd_(—)0879-   MFA: Mfla_(—)2133-   HPY: HP0354(dxs)-   HPJ: jhp0328(dxs)-   HPA: HPAG1_(—)0349-   HHE: HH0608(dxs)-   HAC: Hac_(—)0968(dxs)-   WSU: WS1996-   TDN: Tmden_(—)0475-   CJE: Cj0321(dxs)-   CJR: CJE0366(dxs)-   CJJ: CJJ81176_(—)0343(dxs)-   CJU: C8J_(—)0298(dxs)-   CJD: JJD26997_(—)1642(dxs)-   CFF: CFF8240_(—)0264(dxs)-   CCV: CCV52592_(—)1671(dxs) CCV52592_(—)1722-   CHA: CHAB381_(—)1297(dxs)-   CCO: CCC13826_(—)1594(dxs)-   ABU: Abu_(—)2139(dxs)-   NIS: NIS_(—)0391(dxs)-   SUN: SUN_(—)2055(dxs)-   GSU: GSU0686(dxs-1) GSU1764(dxs-2)-   GME: Gmet_(—)1934 Gmet_(—)2822-   PCA: Pcar_(—)1667-   PPD: Ppro_(—)1191 Ppro_(—)2403-   DVU: DVU1350(dxs)-   DVL: Dvul_(—)1718-   DDE: Dde_(—)2200-   LIP: LI0408(dsx)-   DPS: DP2700-   ADE: Adeh_(—)1097-   MXA: MXAN_(—)4643(dxs)-   SAT: SYN_(—)02456-   SFU: Sfum_(—)1418-   PUB: SAR11_(—)0611(dxs)-   MLO: mlr7474-   MES: Meso_(—)0735-   SME: SMc00972(dxs)-   ATU: Atu0745(dxs)-   ATC: AGR_C_(—)1351-   RET: RHE_CH00913(dxs)-   RLE: RL0973(dxs)-   BME: BMEI1498-   BMF: BAB1_(—)0462(dxs)-   BMS: BR0436(dxs)-   BMB: BruAb1_(—)0458(dxs)-   BOV: BOV_(—)0443(dxs)-   BJA: bll2651(dxs)-   BRA: BRADO2161(dxs)-   BBT: BBta_(—)2479(dxs)-   RPA: RPA0952(dxs)-   RPB: RPB_(—)4460-   RPC: RPC_(—)1149-   RPD: RPD_(—)4305-   RPE: RPE_(—)1067-   NWI: Nwi_(—)0633-   NHA: Nham_(—)0778-   BHE: BH04350(dxs)-   BQU: BQ03540(dxs)-   BBK: BARBAKC583_(—)0400(dxs)-   CCR: CC_(—)2068-   SIL: SP00247(dxs)-   SIT: TM1040_(—)2920-   RSP: RSP_(—)0254(dxsA) RSP_(—)1134(dxs)-   JAN: Jann_(—)0088 Jann_(—)0170-   RDE: RD1_(—)0101(dxs) RD1_(—)0548(dxs)-   MMR: Mmar10_(—)0849-   HNE: HNE_(—)1838(dxs)-   ZMO: ZMO1234(dxs) ZMO1598(dxs)-   NAR: Saro_(—)0161-   SAL: Sala_(—)2354-   ELI: ELI_(—)12520-   GOX: GOX0252-   GBE: GbCGDNIH1_(—)0221 GbCGDNIH1_(—)2404-   RRU: Rru_A0054 Rru_A2619-   MAG: amb2904-   MGM: Mmc1_(—)1048-   SUS: Acid_(—)1783-   BSU: BG11715(dxs)-   BHA: BH2779-   BAN: BA4400(dxs)-   BAR: GBAA4400(dxs)-   BAA: BA_(—)4853-   BAT: BAS4081-   BCE: BC4176(dxs)-   BCA: BCE_(—)4249(dxs)-   BCZ: BCZK3930(dxs)-   BTK: BT9727_(—)3919(dxs)-   BTL: BALH_(—)3785(dxs)-   BLI: BL01523(dxs)-   BLD: BL102598(dxs)-   BCL: ABC2462(dxs)-   BAY: RBAM_(—)022600-   BPU: BPUM_(—)2159-   GKA: GK2392-   GTN: GTNG_(—)2322-   LMO: lmo1365(tktB)-   LMF: LMOf2365_(—)1382(dxs)-   LIN: lin1402(tktB)-   LWE: lwe1380(tktB)-   LLA: L108911(dxsA) L123365(dxsB)-   LLC: LACR_(—)1572 LACR_(—)1843-   LLM: llmg0749(dxsB)-   SAK: SAK_(—)0263-   LPL: lp_(—)2610(dxs)-   LJO: LJ0406-   LAC: LBA0356-   LSL: LSL_(—)0209(dxs)-   LGA: LGAS_(—)0350-   STH: STH1842-   CAC: CAC2077 CA_P0106(dxs)-   CPE: CPE1819-   CPF: CPF_(—)2073(dxs)-   CPR: CPR_(—)1787(dxs)-   CTC: CTC01575-   CNO: NT01CX_(—)1983-   CTH: Cthe_(—)0828-   CDF: CD1207(dxs)-   CBO: CB01881(dxs)-   CBA: CLB_(—)1818(dxs)-   CBH: CLC_(—)1825(dxs)-   CBF: CLI_(—)1945(dxs)-   CKL: CKL_(—)1231(dxs)-   CHY: CHY_(—)1985(dxs)-   DSY: DSY2348-   DRM: Dred_(—)1078-   PTH: PTH_(—)1196(dxs)-   SWO: Swol_(—)0582-   CSC: Csac_(—)1853-   TTE: ITE1298(dxs)-   MTA: Moth_(—)1511-   MPE: MYPE730-   MGA: MGA_(—)1268(dxs)-   MTU: Rv2682c(dxs1) Rv3379c(dxs2)-   MTC: MT2756(dxs)-   MBO: Mb2701c(dxs1) Mb3413c(dxs2)-   MLE: ML1038(dxs)-   MPA: MAP2803c(dxs)-   MAV: MAV_(—)3577(dxs)-   MSM: MSMEG_(—)2776(dxs)-   MMC: Mmcs_(—)2208-   CGL: NCgl1827(cg11902)-   CGB: cg2083(dxs)-   CEF: CE1796-   CDI: DIP1397(dxs)-   CJK: jk1078(dxs)-   NFA: nfa37410(dxs)-   RHA: RHA1_ro06843-   SCO: SCO6013(SC1C3.01) SCO6768(SC6A5.17)-   SMA: SAV1646(dxs1) SAV2244(dxs2)-   TWH: TWT484-   TWS: TW280(Dxs)-   LXX: Lxx10450(dxs)-   CMI: CMM_(—)1660(dxsA)-   AAU: AAur_(—)1790(dxs)-   PAC: PPA1062-   TFU: Tfu_(—)1917-   FRA: Francci3_(—)1326-   FAL: FRAAL2088(dxs)-   ACE: Acel_(—)1393-   SEN: SACE_(—)1815(dxs) SACE_(—)4351-   BLO: BL1132(dxs)-   BAD: BAD_(—)0513(dxs)-   FNU: FN1208 FN1464-   RBA: RB2143(dxs)-   CTR: CT331(dxs)-   CTA: CTA_(—)0359(dxs)-   CMU: TC0608-   CPN: CPn 1060(tktB_(—)2)-   CPA: CP0790-   CPJ: CPj 1060(tktB_(—)2)-   CPT: CpB1102-   CCA: CCA00304(dxs)-   CAB: CAB301(dxs)-   CFE: CF0699(dxs)-   PCU: pc0619(dxs)-   TPA: TP0824-   TDE: TDE1910(dxs)-   LIL: LA3285(dxs)-   LIC: LIC10863(dxs)-   LBJ: LBJ_(—)0917(dxs)-   LBL: LBL_(—)0932(dxs)-   SYN: sl11945(dxs)-   SYW: SYNW1292(Dxs)-   SYC: syc1087_c(dxs)-   SYF: Synpcc7942_(—)0430-   SYD: Syncc9605_(—)1430-   SYE: Syncc9902_(—)1069-   SYG: sync_(—)1410(dxs)-   SYR: SynRCC307_(—)1390(dxs)-   SYX: SynWH7803_(—)1223(dxs)-   CYA: CYA_(—)1701(dxs)-   CYB: CYB_(—)1983(dxs)-   TEL: tl10623-   GVI: gl10194-   ANA: alr0599-   AVA: Ava_(—)4532-   PMA: Pro0928(dxs)-   PMM: PMM0907(Dxs)-   PMT: PMT0685(dxs)-   PMN: PMN2A_(—)0300-   PMI: PMT9312_(—)0893-   PMB: A9601_(—)09541(dxs)-   PMC: P9515_(—)09901(dxs)-   PMF: P9303_(—)15371(dxs)-   PMG: P9301_(—)09521(dxs)-   PMH: P9215_(—)09851-   PMJ: P9211_(—)08521-   PME: NATL1_(—)09721(dxs)-   TER: Tery_(—)3042-   BTH: BT_(—)1403 BT_(—)4099-   BFR: BF0873 BF4306-   BFS: BF0796(dxs) BF4114-   PGI: PG2217(dxs)-   CHU: CHU_(—)3643(dxs)-   GFO: GFO_(—)3470(dxs)-   FPS: FP0279(dxs)-   CTE: CT0337(dxs)-   CPH: Cpha266_(—)0671-   PVI: Cvib_(—)0498-   PLT: Plut_(—)0450-   DET: DET0745(dxs)-   DEH: cbdb_A720(dxs)-   DRA: DR_(—)1475-   DGE: Dgeo_(—)0994-   TTH: TTC1614-   TTJ: TTHA0006-   AAE: aq_(—)881-   TMA: TM1770-   PMO: Pmob_(—)1001

Exemplary Acetyl-CoA-Acetyltransferase Nucleic Acids and Polypeptides

-   HSA: 38(ACAT1) 39(ACAT2)-   PTR: 451528(ACAT1)-   MCC: 707653(ACAT1) 708750(ACAT2)-   MMU: 110446(Acat1) 110460(Acat2)-   RNO: 25014(Acat1)-   CFA: 484063(ACAT2) 489421(ACAT1)-   GGA: 418968(ACAT1) 421587(RCJMB04_(—)34i5)-   XLA: 379569(MGC69098) 414622(MGC81403) 414639(MGC81256)    444457(MGC83664)-   XTR: 394562(acat2)-   DRE: 30643(acat2)-   SPU: 759502(LOC759502)-   DME: Dmel_CG10932 Dmel_CG9149-   CEL: T02G5.4 T02G5.7 T02G5.8(kat-1)-   ATH: AT5G48230(ACAT2/EMB1276)-   OSA: 4326136 4346520-   CME: CMA042C CME087C-   SCE: YPL028W(ERG10)-   AGO: AGOS_ADR165C-   PIC: PICST_(—)31707(ERG10)-   CAL: CaO19.1591(erg10)-   CGR: CAGL0L12364g-   SPO: SPBC215.09c-   MGR: MGG_(—)01755 MGG_(—)13499-   ANI: AN1409.2-   AFM: AFUA_(—)6G14200 AFUA_(—)8G04000-   AOR: AO090103000012 AO090103000406-   CNE: CNC05280-   UMA: UM03571.1-   DDI: DDB_(—)0231621-   PFA: PF14_(—)0484-   TET: TTHERM_(—)00091590 TTHERM00277470 TMERM_(—)00926980-   TCR: 511003.60-   ECO: b2224(atoB)-   ECJ: JW2218(atoB) JW5453(yqeF)-   ECE: Z4164(yqeF)-   ECS: ECs3701-   ECC: c2767(atoB) c3441(yqeF)-   ECI: UTI89_C2506(atoB) UTI89_C3247(yqeF)-   ECP: ECP_(—)2268 ECP_(—)2857-   ECV: APECO1_(—)3662(yqeF) APECO1_(—)4335(atoB) APECO1_(—)43352(atoB)-   ECX: EcHS_A2365-   STY: STY3164(yqeF)-   STT: t2929(yqeF)-   SPT: SPA2886(yqeF)-   SEC: SC2958(yqeF)-   STM: STM3019(yqeF)-   SFL: SF2854(yqeF)-   SFX: S3052(yqeF)-   SFV: SFV_(—)2922(yqeF)-   SSN: SSON_(—)2283(atoB) SSON_(—)3004(yqeF)-   SBO: SBO_(—)2736(yqeF)-   ECA: ECA1282(atoB)-   ENT: Ent638_(—)3299-   SPE: Spro_(—)0592-   HIT: NTHI0932(atoB)-   XCC: XCC1297(atoB)-   XCB: XC_(—)2943-   XCV: XCV1401(thlA)-   XAC: XAC1348(atoB)-   XOO: XOO1881(atoB)-   XOM: XOO_(—)1778(XOO1778)-   VCH: VCA0690-   VCO: VCO395_(—)0630-   VVU: VV2_(—)0494 VV2_(—)0741-   VVY: VVA1043 VVA1210-   VPA: VPA0620 VPA1123 VPA1204-   PPR: PBPRB1112 PBPRB1840-   PAE: PA2001(atoB) PA2553 PA3454 PA3589 PA3925-   PAU: PA14_(—)38630(atoB)-   PPU: PP_(—)2051(atoB) PP_(—)2215(fadAx) PP_(—)3754 PP_(—)4636-   PPF: Pput_(—)2009 Pput_(—)2403 Pput_(—)3523 Pput_(—)4498-   PST: PSPTO_(—)0957(phbA-1) PSPTO_(—)3164(phbA-2)-   PSB: Psyr_(—)0824 Psyr_(—)3031-   PSP: PSPPH0850(phbA1) PSPPH_(—)2209(phbA2)-   PFL: PFL_(—)1478(atoB-2) PFL_(—)2321 PFL_(—)3066 PFL_(—)4330(atoB-2)    PFL_(—)5283-   PFO: Pfl_(—)1269 Pfl_(—)1739 Pfl_(—)2074 Pfl_(—)2868-   PEN: PSEEN3197 PSEEN3547(fadAx) PSEEN4635(phbA)-   PMY: Pmen_(—)1138 Pmen_(—)2036 Pmen_(—)3597 Pmen_(—)3662    Pmen_(—)3820-   PAR: Psyc_(—)0252 Psyc_(—)1169-   PCR: Pcryo_(—)0278 Pcryo_(—)1236 Pcryo_(—)1260-   PRW: PsycPRwf_(—)2011-   ACI: ACIAD0694 ACIAD1612 ACIAD2516(atoB)-   SON: SO_(—)1677(atoB)-   SDN: Sden_(—)1943-   SFR: Sfri_(—)1338 Sfri_(—)2063-   SAZ: Sama_(—)1375-   SBL: Sbal_(—)1495-   SBM: Shew185_(—)1489-   SBN: Sbal195_(—)1525-   SLO: Shew_(—)1667 Shew_(—)2858 SPC: Sputcn32_(—)1397-   SSE: Ssed_(—)1473 Ssed_(—)3533-   SPL: Spea_(—)2783-   SHE: Shewmr4_(—)2597-   SHM: Shewmr7_(—)2664-   SHN: Shewana3_(—)2771-   SHW: Sputw3181_(—)2704-   ILO: IL0872-   CPS: CPS_(—)1605 CPS_(—)2626-   PHA: PSHAa0908 PSHAa1454(atoB) PSHAa1586(atoB)-   PAT: Patl_(—)2923-   SDE: Sde_(—)3149-   PIN: Ping0659 Ping_(—)2401-   MAQ: Maqu_(—)2117 Maqu_(—)2489 Maqu_(—)2696 Maqu_(—)3162-   CBU: CBU_(—)0974-   LPN: lpg1825(atoB)-   LPF: lpl1789-   LPP: lpp 1788-   NOC: Noc_(—)1891-   AEH: Mlg0688 Mlg_(—)2706-   HHA: Hhal_(—)1685-   HCH: HCH_(—)05299-   CSA: Csal_(—)0301 Csal_(—)3068-   ABO: ABO_(—)0648(fadAx)-   MMW: Mmwyl1_(—)0073 Mmwyl1_(—)3021 Mmwyl1_(—)3053 Mmwyl1_(—)3097    Mmwyl1_(—)4182-   AHA: AHA_(—)2143(atoB)-   CVI: CV_(—)2088(atoB) CV_(—)2790(phaA)-   RSO: RSc0276(atoB) RSc 1632(phbA) RSc1637(bktB) RSc1761(RS02948)-   REU: Reut_A0138 Reut_A1348 Reut_A1353 Reut_B4561 Reut_B4738    Reut_B5587 Reut_C5943 Reut_C6062-   REH: H16_A0170 H16_A0867 H16_A0868 H16_A0872 H16_A1297    H16_A1438(phaA) H16_A1445(bktB) H16_A1528 H16_A1713 H16_A1720    H16_A1887 H16_A2148 H16_B0380 H16_B0381 H16_B0406 H16_B0662    H16_B0668 H16_B0759 H16_B1369 H16_B1771-   RME: Rmet_(—)0106 Rmet_(—)1357 Rmet_(—)1362 Rmet_(—)5156-   BMA: BMA1316 BMA1321(phbA) BMA1436-   BMV: BMASAVP1_A1805(bktB) BMASAVP1_A1810(phbA)-   BML: BMA10299_A0086(phbA) BMA10299_A0091-   BMN: BMA10247_(—)1076(bktB) BMA10247_(—)1081(phbA)-   BXE: Bxe_A2273 Bxe_A2335 Bxe_A2342 Bxe_A4255 Bxe_B0377 Bxe_B0739    Bxe_C0332 Bxe_C0574 Bxe_C0915-   BVI: Bcep1808_(—)0519 Bcep1808_(—)1717 Bcep1808_(—)2877    Bcep1808_(—)3594 Bcep1808_(—)4015 Bcep1808_(—)5507 Bcep1808_(—)5644-   BUR: Bcep18194_A3629 Bcep18194_A5080 Bcep18194_A5091 Bcep18194_A6102    Bcep18194_B0263 Bcep18194_B1439 Bcep18194_C6652 Bcep18194_C6802    Bcep18194_C6874 Bcep18194_C7118 Bcep18194_C7151 Bcep18194_C7332-   BCN: Bcen_(—)1553 Bcen_(—)1599 Bcen_(—)2158 Bcen_(—)2563    Bcen_(—)2998 Bcen_(—)6289-   BCH: Bcen2424_(—)0542 Bcen2424_(—)1790 Bcen2424_(—)2772    Bcen2424_(—)5368 Bcen2424_(—)6232 Bcen2424_(—)6276-   BAM: Bamb_(—)0447 Bamb_(—)1728 Bamb_(—)2824 Bamb_(—)4717    Bamb_(—)5771 Bamb_(—)5969-   BPS: BPSL1426 BPSL1535(phbA) BPSL1540-   BPM: BURPS1710b_(—)2325(bktB) BURPS1710b_(—)2330(phbA)    BURPS1710b_(—)2453(atoB-2)-   BPL: BURPS1106A_(—)2197(bktB) BURPS1106A_(—)2202(phbA)-   BPD: BURPS668_(—)2160(bktB) BURPS668_(—)2165(phbA)-   BTE: BTH_(—)12144 BTH_(—)12256 BTH_(—)12261-   PNU: Pnuc_(—)0927-   BPE: BP0447 BP0668 BP2059-   BPA: BPP0608 BPP1744 BPP3805 BPP4216 BPP4361-   BBR: BB0614 BB3364 BB4250 BB4804 BB4947-   RFR: Rfer_(—)0272 Rfer_(—)1000 Rfer_(—)1871 Rfer_(—)2273    Rfer_(—)2561 Rfer_(—)2594 Rfer_(—)3839-   POL: Bpro_(—)1577 Bpro_(—)2140 Bpro_(—)3113 Bpro_(—)4187-   PNA: Pnap_(—)0060 Pnap_(—)0458 Pnap_(—)0867 Pnap_(—)1159    Pnap_(—)2136 Pnap_(—)2804-   AAV: Aave_(—)0031 Aave_(—)2478 Aave_(—)3944 Aave_(—)4368-   AJS: Ajs_(—)0014 Ajs_(—)0124 Ajs_(—)1931 Ajs_(—)2073 Ajs_(—)2317    Ajs_(—)3548-   Ajs_(—)3738 Ajs_(—)3776-   VEI: Veis_(—)1331 Veis_(—)3818 Veis_(—)4193-   DAC: Daci_(—)0025 Daci_(—)0192 Daci_(—)3601 Daci_(—)5988-   MPT: Mpe_A1536 Mpe_A1776 Mpe_A1869 Mpe_A3367-   HAR: HEAR0577(phbA)-   MMS: mma0555-   NEU: NE2262(bktB)-   NET: Neut_(—)0610-   EBA: ebA5202 p2A409(tioL)-   AZO: azo0464(fadAl) azo0469(fadA2) azo2172(thlA)-   DAR: Daro_(—)0098 Daro_(—)3022-   HPA: HPAG10675-   HAC: Hac_(—)0958(atoB)-   GME: Gmet_(—)1719 Gmet_(—)2074 Gmet_(—)2213 Gmet_(—)2268    Gmet_(—)3302-   GUR: Gura_(—)3043-   BBA: Bd0404(atoB) Bd2095-   DOL: Dole_(—)0671 Dole_(—)1778 Dole_(—)2160 Dole_(—)2187-   ADE: Adeh_(—)0062 Adeh_(—)2365-   AFW: Anae109_(—)0064 Anae109_(—)1504-   MXA: MXAN_(—)3791-   SAT: SYN_(—)02642-   SFU: Sfum_(—)2280 Sfum_(—)3582-   RPR: RP737-   RCO: RC1134 RC1135-   RFE: RF_(—)0163(paaJ)-   RBE: RBE_(—)0139(paaJ)-   RAK: A1C_(—)05820-   RBO: A1I_(—)07215-   RCM: A1E_(—)04760-   PUB: SAR11_(—)0428(thlA)-   MLO: mlr3847-   MES: Meso_(—)3374-   PLA: Plav_(—)1573 Plav_(—)2783-   SME: SMa1450 SMc03879(phbA)-   SMD: Smed_(—)0499 Smed_(—)3117 Smed_(—)5094 Smed_(—)5096-   ATU: Atu2769(atoB) Atu3475-   ATC: AGR_C_(—)5022(phbA) AGR_L_(—)2713-   RET: RHE_CH04018(phbAch) RHE_PC00068(ypc00040) RHE_PF00014(phbAf)-   RLE: RL4621(phaA) pRL100301 pRL120369-   BME: BMEIO274 BMEH0817-   BMF: BAB1_(—)1783(phbA-1) BAB2_(—)0790(phbA-2)-   BMS: BR1772(phbA-1) BRA0448(phbA-2)-   BMB: BruAb1_(—)1756(phbA-1) BruAb2_(—)0774(phbA-2)-   BOV: BOV_(—)1707(phbA-1)-   OAN: Oant_(—)1130 Oant_(—)3107 Oant_(—)3718 Oant_(—)4020-   BJA: bll0226(atoB) bll3949 bll7400 bll7819 blr3724(phbA)-   BRA: BRADO0562(phbA) BRADO0983(pimB) BRADO3110 BRADO3134(atoB)-   BBT: BBta_(—)3558 BBta_(—)3575(atoB) BBta_(—)5147(pimB)    BBta_(—)7072(pimB) BBta_(—)7614(phbA)-   RPA: RPA0513(pcaF) RPA0531 RPA3715(pimB)-   RPB: RPB_(—)0509 RPB_(—)0525 RPB_(—)1748-   RPC: RPC_(—)0504 RPC_(—)0636 RPC_(—)0641 RPC_(—)0832 RPC_(—)1050    RPC_(—)2005 RPC_(—)2194 RPC_(—)2228-   RPD: RPD_(—)0306 RPD_(—)0320 RPD_(—)3105 RPD_(—)3306-   RPE: RPE_(—)0168 RPE_(—)0248 RPE_(—)3827-   NWI: Nwi_(—)3060-   XAU: Xaut_(—)3108 Xaut_(—)4665-   CCR: CC_(—)0510 CC_(—)0894 CC_(—)3462-   SIL: SPO0142(bktB) SPO0326(phbA) SPO0773 SPO3408-   SIT: TM1040_(—)0067 TM1040_(—)2790 TM1040_(—)3026 TM1040_(—)3735-   RSP: RSP_(—)0745 RSP_(—)1354 RSP_(—)3184-   RSH: Rsph17029_(—)0022 Rsph17029_(—)2401 Rsph17029_(—)3179    Rsph17029_(—)3921-   RSQ: Rsph17025_(—)0012 Rsph17025_(—)2466 Rsph17025_(—)2833-   JAN: Jann_(—)0262 Jann_(—)0493 Jann_(—)4050-   RDE: RD1_(—)0025 RD1_(—)0201(bktB) RD1_(—)3394(phbA)-   PDE: Pden_(—)2026 Pden_(—)2663 Pden_(—)2870 Pden_(—)2907    Pden_(—)4811 Pden_(—)5022-   DSH: Dshi_(—)0074 Dshi_(—)3066 Dshi_(—)3331-   MMR: Mmar10_(—)0697-   HNE: HNE_(—)2706 HNE_(—)3065 HNE_(—)3133-   NAR: Saro_(—)0809 Saro_(—)1069 Saro_(—)1222 Saro_(—)2306    Saro_(—)2349-   SAL: Sala_(—)0781 Sala_(—)1244 Sala_(—)2896 Sala_(—)3158-   SWI: Swit_(—)0632 Swit_(—)0752 Swit_(—)2893 Swit_(—)3602    Swit_(—)4887 Swit 5019 Swit_(—)5309-   ELI: ELI_(—)01475 ELI_(—)06705 ELI_(—)12035-   GBE: GbCGDNIH1_(—)0447-   ACR: Acry_(—)1847 Acry_(—)2256-   RRU: Rru_A0274 Rru_A1380 Rru_A1469 Rru_A1946 Rru_A3387-   MAG: amb0842-   MGM: Mmc1_(—)1165-   ABA: Acid345_(—)3239-   BSU: BG11319(mmgA) BG13063(yhfS)-   BHA: BH1997 BH2029 BH3801(mmgA)-   BAN: BA3687 BA4240 BA5589-   BAR: GBAA3687 GBAA4240 GBAA5589-   BAA: BA_(—)0445 BA_(—)4172 BA_(—)4700-   BAT: BAS3418 BAS3932 BAS5193-   BCE: BC3627 BC4023 BC5344-   BCA: BCE_(—)3646 BCE_(—)4076 BCE_(—)5475-   BCZ: BCZK3329(mmgA) BCZK3780(thl) BCZK5044(atoB)-   BCY: Bcer98_(—)2722 Bcer98_(—)3865-   BTK: BT9727_(—)3379(mmgA) BT9727_(—)3765(thl) BT9727_(—)5028(atoB)-   BTL: BALH_(—)3262(mmgA) BALH_(—)3642(fadA) BALH_(—)4843(atoB)-   BLI: BL03925(mmgA)-   BLD: BLi03968(mmgA)-   BCL: ABC0345 ABC2989 ABC3617 ABC3891(mmgA)-   BAY: RBAMO22450-   BPU: BPUM_(—)2374(yhfS) BPUM_(—)2941 BPUM_(—)3373-   OIH: OB06760B06890B26320B3013-   GKA: GK1658 GK3397-   SAU: SA0342 SA0534(vraB)-   SAV: SAV0354 SAV0576(vraB)-   SAM: MWO330 MW0531(vraB)-   SAR: SAR0351(thl) SAR0581-   SAS: SAS0330 SAS0534-   SAC: SACOL0426 SACOL0622(atoB)-   SAB: SAB0304(thl) SAB0526-   SAA: SAUSA300_(—)0355 SAUSA300_(—)0560(vraB)-   SAO: SAOUHSC_(—)00336 SAOUHSC_(—)00558-   SAJ: SaurJH9_(—)0402-   SAH: SaurJH1_(—)0412-   SEP: SE0346 SE2384-   SER: SERP0032 SERP0220-   SHA: SH0510(mvaC) SH2417-   SSP: SSP0325 SSP2145-   LMO: lmo 1414-   LMF: LMOf2365_(—)1433-   LIN: lin1453-   LWE: lwe1431-   LLA: L11745(thiL) L25946(fadA)-   LLC: LACR_(—)1665 LACR_(—)1956-   LLM: llmg_(—)0930(thiL)-   SPY: SPy_(—)0140 SPy_(—)1637(atoB)-   SPZ: M5005_Spy_(—)0119 M5005_Spy_(—)0432 M5005_Spy_(—)1344(atoB)-   SPM: spyM18_(—)0136 spyM18_(—)1645(atoB)-   SPG: SpyM3_(—)0108 SpyM3_(—)1378(atoB)-   SPS: SPs0110 SPs0484-   SPH: MGAS10270_Spy0121 MGAS10270_Spy0433 MGAS10270_Spy1461(atoB)-   SPI: MGAS10750_Spy0124 MGAS10750_Spy0452 MGAS10750_Spy1453(atoB)-   SPJ: MGAS2096_Spy0123 MGAS2096_Spy0451 MGAS2096_Spy1365(atoB)-   SPK: MGAS9429_Spy0121 MGAS9429_Spy0431 MGAS9429_Spy1339(atoB)-   SPF: SpyM50447(atoB2)-   SPA: M6_Spy0166 M6_Spy0466 M6_Spy1390-   SPB: M28_Spy0117 M28_Spy0420 M28_Spy1385(atoB)-   SAK: SAK_(—)0568-   LJO: 111609-   LAC: LBA0626(thiL)-   LSA: LSA1486-   LDB: Ldb0879-   LBU: LBUL_(—)0804-   LBR: LVIS_(—)2218-   LCA: LSEI_(—)1787-   LGA: LGAS_(—)1374-   LRE: Lreu_(—)0052-   EFA: EF1364-   OOE: OEOE_(—)0529-   STH: STH2913 STH725 STH804-   CAC: CAC2873 CA_P0078(thiL)-   CPE: CPE2195(atoB)-   CPF: CPF_(—)2460-   CPR: CPR_(—)2170-   CTC: CTC00312-   CNO: NT01CX_(—)0538 NT01CX_(—)0603-   CDF: CD1059(thlA1) CD2676(thlA2)-   CBO: CB03200(thl)-   CBE: Cbei_(—)0411 Cbei_(—)3630-   CKL: CKL_(—)3696(thlA1) CKL_(—)3697(thlA2) CKL_(—)3698(thlA3)-   AMT: Amet_(—)4630-   AOE: Clos0084 Clos0258-   CHY: CHY_(—)1288 CHY_(—)1355(atoB) CHY_(—)1604 CHY_(—)1738-   DSY: DSY0632 DSY0639 DSY1567 DSY1710 DSY2402 DSY3302-   DRM: Dred_(—)0400 Dred_(—)1491 Dred_(—)1784 Dred_(—)1892-   SWO: Swol_(—)0308 Swol_(—)0675 Swol_(—)0789 Swol_(—)1486    Swol_(—)1934 Swol_(—)2051-   TTE: TTE0549(paaJ)-   MTA: Moth_(—)1260-   MTU: Rv1135A Rv1323(fadA4) Rv3546(fadA5)-   MTC: MT1365(phbA)-   MBO: Mb1167 Mb1358(fadA4) Mb3576(fadA5) Mb3586c(fadA6)-   MBB: BCG_(—)1197 BCG_(—)1385(fadA4) BCG_(—)3610(fadA5)    BCG_(—)3620c(fadA6)-   MLE: ML1158(fadA4)-   MPA: MAP2407c(fadA3) MAP2436c(fadA4)-   MAV: MAV_(—)1544 MAV_(—)1573 MAV_(—)1863 MAV_(—)5081-   MSM: MSMEG_(—)2224 MSMEG_(—)4920-   MUL: MUL_(—)0357-   MVA: Mvan_(—)1976 Mvan_(—)1988 Mvan_(—)4305 Mvan_(—)4677    Mvan_(—)4891-   MGI: Mflv_(—)1347 Mflv_(—)1484 Mflv_(—)2040 Mflv_(—)2340    Mflv_(—)4356 Mflv_(—)4368-   MMC: Mmcs_(—)1758 Mmcs_(—)1769 Mmcs_(—)3796 Mmcs_(—)3864-   MKM: Mkms_(—)0251 Mkms_(—)1540 Mkms_(—)1805 Mkms_(—)1816    Mkms_(—)2836 Mkms_(—)3159 Mkms_(—)3286 Mkcms_(—)3869 Mkms_(—)3938    Mkms_(—)4227 Mkms_(—)4411 Mkms_(—)4580 Mkms_(—)4724 Mkms_(—)4764    Mkms_(—)4776-   MJL: Mjls_(—)0231 Mjls_(—)1739 Mjls_(—)1750 Mjls_(—)2819    Mjls_(—)3119 Mjls_(—)3235 Mjls_(—)3800 Mjls_(—)3850 Mjls_(—)4110    Mjls_(—)4383 Mjls_(—)4705 Mjls_(—)4876 Mjls_(—)5018 Mjls_(—)5063    Mjls_(—)5075-   CGL: NCg12309(cg12392)-   CGB: cg2625(pcaF)-   CEF: CE0731 CE2295-   CJK: jk1543(fadA3)-   NFA: nfal0750(fadA4)-   RHA: RHA1_ro01455 RHA1_ro01623 RHA1_ro01876 RHA1_ro02517(catF)    RHA1_ro03022 RHA1_ro03024 RHA1_ro03391 RHA1_ro03892 RHA1_ro04599    RHA1_ro05257 RHA1_ro08871-   SCO: SCO5399(SC8F4.03)-   SMA: SAV1384(fadA5) SAV2856(fadAl)-   ART: Arth_(—)1160 Arth_(—)2986 Arth_(—)3268 Arth_(—)4073-   NCA: Noca_(—)1371 Noca_(—)1797 Noca_(—)1828 Noca_(—)2764    Noca_(—)4142-   TFU: Tfu_(—)1520 Tfu_(—)2394-   FRA: Francci3_(—)3687-   FRE: Franean1_(—)1044 Franean1_(—)2711 Franean1_(—)2726    Franean1_(—)3929 Franean1_(—)4037 Franean14577-   FAL: FRAAL2514 FRAAL2618 FRAAL5910(atoB)-   ACE: Acel_(—)0626 Acel_(—)0672-   SEN: SACE_(—)1192(mmgA) SACE_(—)2736(fadA6) SACE_(—)4011(catF)    SACE_(—)6236(fadA4)-   STP: Strop_(—)3610-   SAQ: Sare_(—)1316 Sare_(—)3991-   RXY: Rxyl_(—)1582 Rxyl_(—)1842 Rxyl_(—)2389 Rxyl_(—)2530-   FNU: FN0495-   BGA: BG0110(fadA)-   BAF: BAPKO_(—)0110(fadA)-   LIL: LA0457(thiL1) LA0828(thiL2) LA4139(fadA)-   LIC: LIC10396(phbA)-   LBJ: LBJ_(—)2862(paaJ-4)-   LBL: LBL_(—)0209(paaJ-4)-   SYN: slr1993(phaA)-   SRU: SRU_(—)1211(atoB) SRU_(—)1547-   CHU: CHU_(—)1910(atoB)-   GFO: GFO_(—)1507(atoB)-   FJO: Fjoh_(—)4612-   FPS: FP0770 FP1586 FP1725-   RRS: RoseRS_(—)3911 RoseRS_(—)4348-   RCA: Rcas_(—)0702 Rcas_(—)3206-   HAU: Haur_(—)0522-   DRA: DR_(—)1072 DR_(—)1428 DR_(—)1960 DR_(—)2480 DR_A0053-   DGE: Dgeo_(—)0755 Dgeo_(—)1305 Dgeo_(—)1441 Dgeo_(—)1883-   TTH: TTC0191 TTC0330-   TTJ: TTHA0559-   TME: Tmel_(—)1134-   FNO: Fnod0314-   PMO: Pmob_(—)0515-   HMA: rrnAC0896(acaB3) rrnAC2815(aca2) rrnAC3497(yqeF) rrnB0240(aca1)    rrnB0242(acaB2) rrnB0309(acaB1)-   TAC: Ta0582-   TVO: TVN0649-   PTO: PT01505-   APE: APE_(—)2108-   SSO: SSO2377(acaB-4)-   STO: ST0514-   SAI: Saci_(—)0963 Saci_(—)1361(acaB1)-   MSE: Msed_(—)0656-   PAI: PAE1220-   PIS: Pisl_(—)0029 Pisl_(—)1301-   PCL: Pcal_(—)0781-   PAS: Pars_(—)0309 Pars_(—)1071-   CMA: Cmaq_(—)1941

Exemplary HMG-CoA Synthase Nucleic Acids and Polypeptides

-   HSA: 3157(HMGCS1) 3158(HMGCS2)-   PTR: 457169(HMGCS2) 461892(HMGCS1)-   MCC: 702553(HMGCS1) 713541(HMGCS2)-   MMU: 15360(Hmgcs2) 208715(Hmgcs1)-   RNO: 24450(Hmgcs2) 29637(Hmgcs1)-   CFA: 479344(HMGCS1) 607923(HMGCS2)-   BTA: 407767(HMGCS1)-   SSC: 397673(CH242-38B5.1)-   GGA: 396379(HMGCS1)-   XLA: 380091(hmgcs1) 447204(MGC80816)-   DRE: 394060(hmgcs1)-   SPU: 578259(L00578259)-   DME: Dmel_CG4311(Hmgs)-   CEL: F25B4.6-   ATH: AT4G11820(BAP1)-   OSA: 4331418 4347614-   CME: CMM189C-   SCE: YML126C(ERG13)-   AGO: AGOS_ADL356C-   PIC: PICST_(—)83020-   CAL: CaO19_(—)7312(CaO19.7312)-   CGR: CAGL0H04081g-   SPO: SPAC4F8.14c(hcs)-   MGR: MGG_(—)01026-   ANI: AN4923.2-   AFM: AFUA_(—)3G10660 AFUA_(—)8G07210-   AOR: AO0003000611 AO090010000487-   CNE: CNC05080 CNG02670-   UMA: UM05362.1-   ECU: ECU10_(—)0510-   DDI: DDBDRAFT_(—)0217522 DDB_(—)0219924(hgsA)-   TET: TTHERM_(—)00691190-   TBR: Tb927.8.6110-   YPE: YP01457-   YPK: y2712(pksG)-   YPM: YP_(—)1349(pksG)-   YPA: YPA_(—)0750-   YPN: YPN_(—)2521-   YPP: YPDSF_(—)1517-   YPS: YPTB1475-   CBD: COXBU7E912_(—)1931-   TCX: Tcr_(—)1719-   DNO: DNO_(—)0799-   BMA: BMAAl212-   BPS: BPSS1002-   BPM: BURPS1710b_A2613-   BPL: BURPS1106A_A1384-   BPD: BURPS668_A1470-   BTE: BTH_II1670-   MXA: MXAN_(—)3948(tac) MXAN_(—)4267(mvaS)-   BSU: BG10926(pksG)-   OIH: OB2248-   SAU: SA2334(mvaS)-   SAV: SAV2546(mvaS)-   SAM: MW2467(mvaS)-   SAR: SAR2626(mvaS)-   SAS: SAS2432-   SAC: SACOL2561-   SAB: SAB2420(mvaS)-   SAA: SAUSA300_(—)2484-   SAO: SAOUHSC_(—)02860-   SAJ: SaurJH9_(—)2569-   SAH: SaurJH1_(—)2622-   SEP: SE2110-   SER: SERP2122-   SHA: SH0508(mvaS)-   SSP: SSP0324-   LMO: lmo1415-   LMF: LMOf2365_(—)1434(mvaS)-   LIN: lin1454-   LWE: lwe1432(mvaS)-   LLA: L13187(hmcM)-   LLC: LACR_(—)1666-   LLM: llmg_(—)0929(hmcM)-   SPY: SPy_(—)0881(mvaS.2)-   SPZ: M5005_Spy0687(mvaS.1)-   SPM: spyM18_(—)0942(mvaS2)-   SPG: SpyM3_(—)0600(mvaS.2)-   SPS: SPs1253-   SPH: MGAS10270_Spy0745(mvaS1)-   SPI: MGAS10750_Spy0779(mvaS1)-   SPJ: MGAS2096_Spy0759(mvaS1)-   SPK: MGAS9429_Spy0743(mvaS1)-   SPF: SpyM51121(mvaS)-   SPA: M6_Spy0704-   SPB: M28_Spy0667(mvaS.1)-   SPN: SP_(—)1727-   SPR: spr1571(mvaS)-   SPD: SPD_(—)1537(mvaS)-   SAG: SAG1316-   SAN: gbs1386-   SAK: SAK_(—)1347-   SMU: SMU.943c-   STC: str0577(mvaS)-   STL: stu0577(mvaS)-   STE: STER_(—)0621-   SSA: SSA_(—)0338(mvaS)-   SSU: SSU05_(—)1641-   SSV: SSU98_(—)1652-   SGO: SGO_(—)0244-   LPL: lp_(—)2067(mvaS)-   LJO: LJ1607-   LAC: LBA0628(hmcS)-   LSA: LSA1484(mvaS)-   LSL: LSL_(—)0526-   LDB: Ldb0881(mvaS)-   LBU: LBUL_(—)0806-   LBR: LVIS_(—)1363-   LCA: LSEI_(—)1785-   LGA: LGAS_(—)1372-   LRE: Lreu_(—)0676-   PPE: PEPE_(—)0868-   EFA: EF1363-   OOE: OEOE0968-   LME: LEUM_(—)1184-   NFA: nfa22120-   SEN: SACE_(—)4570(pksG)-   BBU: BB0683-   BGA: BG0706-   BAF: BAPKO_(—)0727-   FJO: Fjoh_(—)0678-   HAL: VNG1615G(mvaB)-   HMA: rrnAC1740(mvaS)-   HWA: HQ2868A(mvaB)-   NPH: NP2608A(mvaB_(—)1) NP4836A(mvaB_(—)2)

Exemplary Hydroxymethylglutaryl-CoA Reductase Nucleic Acids andPolypeptides

-   HSA: 3156(HMGCR)-   PTR: 471516(HMGCR)-   MCC: 705479(HMGCR)-   MMU: 15357(Hmgcr)-   RNO: 25675(Hmgcr)-   CFA: 479182(HMGCR)-   BTA: 407159(HMGCR)-   GGA: 395145(RCJMB04_(—)14 m24)-   SPU: 373355(LOC373355)-   DME: Dmel_CG10367(Hmgcr)-   CEL: F08F8.2-   OSA: 4347443-   SCE: YLR450W(HMG2) YML075C(HMG1)-   AGO: AGOS_AER152W-   CGR: CAGLOL11506g-   SPO: SPCC162.09c(hmg1)-   ANI: AN3817.2-   AFM: AFUA_(—)1G11230 AFUA_(—)2G03700-   AOR: AO090103000311 AO090120000217-   CNE: CNF04830-   UMA: UM03014.1-   ECU: ECU10_(—)1720-   DDI: DDB_(—)0191125(hmgA) DDB_(—)0215357(hmgB)-   TBR: Tb927.6.4540-   TCR: 506831.40 509167.20-   LMA: LmjF30.3190-   VCH: VCA0723-   VCO: VC0395_(—)0662-   VVU: VV2_(—)0117-   VVY: VVA0625-   VPA: VPA0968-   VFI: VFA0841-   PAT: Patl_(—)0427-   CBU: CBU_(—)0030 CBU_(—)0610-   CBD: COXBU7E912_(—)0151 COXBU7E912_(—)0622(hmgA)-   TCX: Tcr_(—)1717-   DNO: DNO_(—)0797-   CVI: CV_(—)1806-   SUS: Acid_(—)5728 Acid_(—)6132-   SAU: SA2333(mvaA)-   SAV: SAV2545(mvaA)-   SAM: MW2466(mvaA)-   SAB: SAB2419c(mvaA)-   SEP: SE2109-   LWE: lwe0819(mvaA)-   LLA: L10433(mvaA)-   LLC: LACR_(—)1664-   LLM: llmg_(—)0931(mvaA)-   SPY: SPy_(—)0880(mvaS.1)-   SPM: spyM18_(—)0941(mvaS1)-   SPG: SpyM3_(—)0599(mvaS.1)-   SPS: SPs1254-   SPH: MGAS10270_Spy0744-   SPI: MGAS10750_Spy0778-   SPJ: MGAS2096_Spy0758-   SPK: MGAS9429_Spy0742-   SPA: M6_Spy0703-   SPN: SP_(—)1726-   SAG: SAG1317-   SAN: gbs1387-   STC: str0576(mvaA)-   STL: stu0576(mvaA)-   STE: STER_(—)0620-   SSA: SSA_(—)0337(mvaA)-   LPL: lp_(—)0447(mvaA)-   LJO: LJ1608-   LSL: LSL_(—)0224-   LBR: LVIS_(—)0450-   LGA: LGAS_(—)1373-   EFA: EF1364-   NFA: nfa22110-   BGA: BG0708(mvaA)-   SRU: SRU_(—)2422-   FPS: FP2341-   MMP: MMP0087(hmgA)-   MMQ: MmarC5_(—)1589-   MAC: MA3073(hmgA)-   MBA: Mbar_A1972-   MMA: MM_(—)0335-   MBU: Mbur_(—)1098-   MHU: Mhun_(—)3004-   MEM: Memar_(—)2365-   MBN: Mboo_(—)0137-   MTH: MTH562-   MST: Msp_(—)0584(hmgA)-   MSI: Msm_(—)0227-   MKA: MK0355(HMG1)-   AFU: AF1736(mvaA)-   HAL: VNG1875G(mvaA)-   HMA: rrnAC3412(mvaA)-   HWA: HQ3215A(hmgR)-   NPH: NP0368A(mvaA_(—)2) NP2422A(mvaA_(—)1)-   TAC: Ta0406m-   TVO: TVN1168-   PTO: PT01143-   PAB: PAB2106(mvaA)-   PFU: PF1848-   TKO: TK0914-   RCI: RCIX1027(hmgA) RCIX376(hmgA)-   APE: APE_(—)1869-   IHO: Igni_(—)0476-   HBU: Hbut_(—)1531-   SSO: SS00531-   STO: ST1352-   SAI: Saci_(—)1359-   PAI: PAE2182-   PIS: Pisl_(—)0814-   PCL: Pcal_(—)1085-   PAS: Pars_(—)0796

Exemplary Mevalonate Kinase Nucleic Acids and Polypeptides

-   HSA: 4598(MVK)-   MCC: 707645(MVK)-   MMU: 17855(Mvk)-   RNO: 81727(Mvk)-   CFA: 486309(MVK)-   BTA: 505792(MVK)-   GGA: 768555(MVK)-   DRE: 492477(zgc:103473)-   SPU: 585785(LOC585785)-   DME: Dmel_CG33671-   OSA: 4348331-   SCE: YMR208W(ERG12)-   AGO: AGOS_AER335W-   PIC: PICST_(—)40742(ERG12)-   CGR: CAGLOF03861g-   SPO: SPAC13G6.11c-   MGR: MGG_(—)06946-   ANI: AN3869.2-   AFM: AFUA_(—)4G07780-   AOR: AO090023000793-   CNE: CNK01740-   ECU: ECU09_(—)1780-   DDI: DDBDRAFT_(—)0168621-   TET: TTHERM_(—)00637680-   TBR: Tb927.4.4070-   TCR: 436521.9 509237.10-   LMA: LmjF31.0560-   CBU: CBU_(—)0608 CBU_(—)0609-   CBD: COXBU7E912_(—)0620(mvk)-   LPN: lpg2039-   LPF: lpl2017-   LPP: lpp 2022-   BBA: Bd1027(1 mbP) Bd1630(mvk)-   MXA: MXAN_(—)5019(mvk)-   OIH: OB0225-   SAU: SA0547(mvaK1)-   SAV: SAV0590(mvaK1)-   SAM: MW0545(mvaK1)-   SAR: SAR0596(mvaK1)-   SAS: SAS0549-   SAC: SACOL0636(mvk)-   SAB: SAB0540(mvaK1)-   SAA: SAUSA300_(—)0572(mvk)-   SAO: SAOUHSC_(—)00577-   SEP: SE0361-   SER: SERP0238(mvk)-   SHA: SH2402(mvaK1)-   SSP: SSP2122-   LMO: lmo0010-   LMF: LMOf2365_(—)0011-   LIN: lin0010-   LWE: lwe0011(mvk)-   LLA: L7866(yeaG)-   LLC: LACR_(—)0454-   LLM: llmg_(—)0425(mvk)-   SPY: SPy_(—)0876(mvaK1)-   SPZ: M5005_Spy_(—)0682(mvaK1)-   SPM: spyM18_(—)0937(mvaK1)-   SPG: SpyM3_(—)0595(mvaK1)-   SPS: SPs1258-   SPH: MGAS10270_Spy0740(mvaK1)-   SPI: MGAS10750_Spy0774(mvaK1)-   SPJ: MGAS2096_Spy0753(mvaK1)-   SPK: MGAS9429_Spy0737(mvaK1)-   SPF: SpyM51126(mvaK1)-   SPA: M6_Spy0699-   SPB: M28_Spy0662(mvaK1)-   SPN: SP_(—)0381-   SPR: spr0338(mvk)-   SPD: SPD_(—)0346(mvk)-   SAG: SAG1326-   SAN: gbs1396-   SAK: SAK_(—)1357(mvk)-   SMU: SMU.181-   STC: str0559(mvaK1)-   STL: stu0559(mvaK1)-   STE: STER_(—)0598-   SSA: SSA_(—)0333(mvaK1)-   SSU: SSU05_(—)0289-   SSV: SSU980285-   SGO: SGO_(—)0239(mvk)-   LPL: lp_(—)1735(mvaK1)-   LJO: LJ1205-   LAC: LBA1167(mvaK)-   LSA: LSA0908(mvaK1)-   LSL: LSL_(—)0685(eRG)-   LDB: Ldb0999(mvk)-   LBU: LBUL_(—)0906-   LBR: LVIS_(—)0858-   LCA: LSEI_(—)1491-   LGA: LGAS_(—)1033-   LRE: Lreu_(—)0915-   PPE: PEPE_(—)0927-   EFA: EF0904(mvk)-   OOE: OEOE_(—)1100-   LME: LEUM_(—)1385-   NFA: nfa22070-   BGA: BG0711-   BAF: BAPKO_(—)0732-   FPS: FP0313-   MMP: MMP1335-   MAE: Maeo_(—)0775-   MAC: MA0602(mvk)-   MBA: Mbar_A1421-   MMA: MM_(—)1762-   MBU: Mbur_(—)2395-   MHU: Mhun_(—)2890-   MEM: Memar_(—)1812-   MBN: Mboo_(—)2213-   MST: Msp_(—)0858(mvk)-   MSI: Msm_(—)1439-   MKA: MK0993(ERG12)-   HAL: VNG1145G(mvk)-   HMA: rrnAC0077(mvk)-   HWA: HQ2925A(mvk)-   NPH: NP2850A(mvk)-   PTO: PT01352-   PHO: PH1625-   PAB: PAB0372(mvk)-   PFU: PF1637(mvk)-   TKO: TK1474-   RCI: LRC399(mvk)-   APE: APE_(—)2439-   HBU: Hbut_(—)0877-   SSO: SS00383-   STO: ST2185-   SAI: Saci_(—)2365(mvk)-   MSE: Msed_(—)1602-   PAI: PAE3108-   PIS: Pisl_(—)0467-   PCL: Pcal_(—)1835

Exemplary Phosphomevalonate Kinase Nucleic Acids and Polypeptides

-   HSA: 10654(PMVK)-   PTR: 457350(PMVK)-   MCC: 717014(PMVK)-   MMU: 68603(Pmvk)-   CFA: 612251(PMVK)-   BTA: 513533(PMVK)-   DME: Dmel_CG10268-   ATH: AT1G31910-   OSA: 4332275-   SCE: YMR220W(ERG8)-   AGO: AGOS_AER354W-   PIC: PICST_(—)52257(ERG8)-   CGR: CAGL0F03993g-   SPO: SPAC343.01c-   MGR: MGG_(—)05812-   ANI: AN2311.2-   AFM: AFUA_(—)5G10680-   AOR: AO090010000471-   CNE: CNM00100-   UMA: UM00760.1-   DDI: DDBDRAFT_(—)0184512-   TBR: Tb09.160.3690-   TCR: 507913.20 508277.140-   LMA: LmjF15.1460-   MXA: MXAN_(—)5017-   OIH: OB0227-   SAU: SA0549(mvaK2)-   SAV: SAV0592(mvaK2)-   SAM: MWO547(mvaK2)-   SAR: SAR0598(mvaK2)-   SAS: SAS0551-   SAC: SACOL0638-   SAB: SAB0542(mvaK2)-   SAA: SAUSA300_(—)0574-   SAO: SAOUHSC_(—)00579-   SAJ: SauJH9_(—)0615-   SEP: SE0363-   SER: SERP0240-   SHA: SH2400(mvaK2)-   SSP: SSP2120-   LMO: lmo0012-   LMF: LMOf2365_(—)0013-   LIN: lin0012-   LWE: lwe0013-   LLA: L10014(yebA)-   LLC: LACR_(—)0456-   LLM: llmg0427-   SPY: SPy_(—)0878(mvaK2)-   SPZ: M5005_Spy_(—)0684(mvaK2)-   SPM: spyM18_(—)0939-   SPG: SpyM3_(—)0597(mvaK2)-   SPS: SPs1256-   SPH: MGAS10270_Spy0742(mvaK2)-   SPI: MGAS10750_Spy0776(mvaK2)-   SPJ: MGAS2096_Spy0755(mvaK2)-   SPK: MGAS9429_Spy0739(mvaK2)-   SPF: SpyM51124(mvaK2)-   SPA: M6_Spy0701-   SPB: M28_Spy0664(mvaK2)-   SPN: SP_(—)0383-   SPR: spr0340(mvaK2)-   SPD: SPD_(—)0348(mvaK2)-   SAG: SAG1324-   SAN: gbs1394-   SAK: SAK_(—)1355-   SMU: SMU.938-   STC: str0561(mvaK2)-   STL: stu0561(mvaK2)-   STE: STER_(—)0600-   SSA: SSA_(—)0335(mvaK2)-   SSU: SSU050291-   SSV: SSU98_(—)0287-   SGO: SGO_(—)0241-   LPL: lp_(—)1733(mvaK2)-   LJO: LJ1207-   LAC: LBA1169-   LSA: LSA0906(mvaK2)-   LSL: LSL_(—)0683-   LDB: Ldb0997(mvaK)-   LBU: LBUL0904-   LBR: LVIS_(—)0860-   LCA: LSEI_(—)1092-   LGA: LGAS_(—)1035-   LRE: Lreu_(—)0913-   PPE: PEPE_(—)0925-   EFA: EF0902-   NFA: nfa22090-   BGA: BG0710-   BAF: BAPKO_(—)0731-   NPH: NP2852A-   SSO: SSO2988-   STO: ST0978-   SAI: Saci_(—)1244

Exemplary Diphosphomevalonate Decarboxylase Nucleic Acids andPolypeptides

-   HSA: 4597(MVD)-   PTR: 468069(MVD)-   MCC: 696865(MVD)-   MMU: 192156(Mvd)-   RNO: 81726(Mvd)-   CFA: 489663(MVD)-   GGA: 425359(MVD)-   DME: Dmel_CG8239-   SCE: YNR043W(MVD1)-   AGO: AGOS_AGL232C-   PIC: PICST_(—)90752-   CGR: CAGL0C03630g-   SPO: SPAC24C9.03-   MGR: MGG_(—)09750-   ANI: AN4414.2-   AFM: AFUA_(—)4G07130-   AOR: AO090023000862-   CNE: CNL04950-   UMA: UM05179.1-   DDI: DDBDRAFT_(—)0218058-   TET: TTHERM_(—)00849200-   TBR: Tb10.05.0010 Tb10.61.2745-   TCR: 507993.330 511281.40-   LMA: LmjF18.0020-   CBU: CBU_(—)0607(mvaD)-   CBD: COXBU7E912_(—)0619(mvaD)-   LPN: lpg2040-   LPF: lpl2018-   LPP: lpp2023-   TCX: Tcr_(—)1734-   DNO: DNO_(—)0504(mvaD)-   BBA: Bd1629-   MXA: MXAN_(—)5018(mvaD)-   OIH: OB0226-   SAU: SA0548(mvaD)-   SAV: SAV0591(mvaD)-   SAM: MWO546(mvaD)-   SAR: SAR0597(mvaD)-   SAS: SAS0550-   SAC: SACOL0637(mvaD)-   SAB: SAB0541(mvaD)-   SAA: SAUSA300_(—)0573(mvaD)-   SAO: SAOUHSC_(—)00578-   SAJ: SaurJH9_(—)0614-   SAH: SaurJH1_(—)0629-   SEP: SE0362-   SER: SERP0239(mvaD)-   SHA: SH2401(mvaD)-   SSP: SSP2121-   LMO: lmo0011-   LMF: LMOf2365_(—)0012(mvaD)-   LIN: lin0011-   LWE: lwe0012(mvaD)-   LLA: L9089(yeaH)-   LLC: LACR_(—)0455-   LLM: llmg0426(mvaD)-   SPY: SPy_(—)0877(mvaD)-   SPZ: M5005_Spy_(—)0683(mvaD)-   SPM: spyM18_(—)0938(mvd)-   SPG: SpyM3_(—)0596(mvaD)-   SPS: SPs1257-   SPH: MGAS10270_Spy0741(mvaD)-   SPI: MGAS10750_Spy0775(mvaD)-   SPJ: MGAS2096_Spy0754(mvaD)-   SPK: MGAS9429_Spy0738(mvaD)-   SPF: SpyM51125(mvaD)-   SPA: M6_Spy0700-   SPB: M28_Spy0663(mvaD)-   SPN: SP_(—)0382-   SPR: spr0339(mvd1)-   SPD: SPD_(—)0347(mvaD)-   SAG: SAG1325(mvaD)-   SAN: gbs1395-   SAK: SAK_(—)1356(mvaD)-   SMU: SMU.937-   STC: str0560(mvaD)-   STL: stu0560(mvaD)-   STE: STER_(—)0599-   SSA: SSA_(—)0334(mvaD)-   SSU: SSU05_(—)0290-   SSV: SSU98_(—)0286-   SGO: SGO_(—)0240(mvaD)-   LPL: lp_(—)1734(mvaD)-   LJO: LJ1206-   LAC: LBA1168(mvaD)-   LSA: LSA0907(mvaD)-   LSL: LSL_(—)0684-   LDB: Ldb0998(mvaD)-   LBU: LBUL_(—)0905-   LBR: LVIS_(—)0859-   LCA: LSEI_(—)1492-   LGA: LGAS_(—)1034-   LRE: Lreu_(—)0914-   PPE: PEPE_(—)0926-   EFA: EF0903(mvaD)-   LME: LEUM_(—)1386-   NFA: nfa22080-   BBU: BB0686-   BGA: BG0709-   BAF: BAPKO0730-   GFO: GFO_(—)3632-   FPS: FP0310(mvaD)-   HAU: Haur_(—)1612-   HAL: VNG0593G(dmd)-   HMA: rrnAC1489(dmd)-   HWA: HQ1525A(mvaD)-   NPH: NP1580A(mvaD)-   PTO: PT00478 PT01356-   SSO: SSO2989-   STO: ST0977-   SAI: Saci_(—)1245(mvd)-   MSE: Msed_(—)1576

Exemplary Isopentenyl Phosphate Kinases (IPK) Nucleic Acids andPolypeptides

-   Methanobacterium thermoautotrophicum gi|2621082-   Methanococcus jannaschii DSM 2661 gi|1590842;-   Methanocaldococcus jannaschii gi|1590842-   Methanothermobacter thermautotrophicus gi|2621082-   Picrophilus torridus DSM9790 (IG-57) gi|48477569-   Pyrococcus abyssi gi|14520758-   Pyrococcus horikoshii OT3 gi|3258052-   Archaeoglobus fulgidus DSM4304 gi|2648231

Exemplary Isopentenyl-Diphosphate Delta-Isomerase (IDI) Nucleic Acidsand Polypeptides

-   HSA: 3422(IDI1) 91734(IDI2)-   PTR: 450262(ID12) 450263(IDI1)-   MCC: 710052(LOC710052) 721730(LOC721730)-   MMU: 319554(Idi1)-   RNO: 89784(Idi1)-   GGA: 420459(IDI1)-   XLA: 494671(LOC494671)-   XTR: 496783(idi2)-   SPU: 586184(LOC586184)-   CEL: K06H7.9(idi-1)-   ATH: AT3G02780(IPP2)-   OSA: 4338791 4343523-   CME: CMB062C-   SCE: YPL117C(IDI1)-   AGO: AGOS_ADL268C-   PIC: PICST_(—)68990(IDI1)-   CGR: CAGL0J06952g-   SPO: SPBC106.15(idi1)-   ANI: AN0579.2-   AFM: AFUA_(—)6G11160-   AOR: AO090023000500-   CNE: CNA02550-   UMA: UM04838.1-   ECU: ECU02_(—)0230-   DDI: DDB_(—)0191342(ipi)-   TET: TTHERM_(—)00237280 TTHERM_(—)00438860-   TBR: Tb09.211.0700-   TCR: 408799.19 510431.10-   LMA: LmjF35.5330-   EHI: 46.t00025-   ECO: b2889(idi)-   ECJ: JW2857(idi)-   ECE: Z4227-   ECS: ECs3761-   ECC: c3467-   ECI: UTI89_C3274-   ECP: ECP_(—)2882-   ECV: APECO1_(—)3638-   ECW: EcE24377A_(—)3215(idi)-   ECX: EcHS_A3048-   STY: STY3195-   STT: t2957-   SPT: SPA2907(idi)-   SEC: SC2979(idi)-   STM: STM3039(idi)-   SFL: SF2875(idi)-   SFX: S3074-   SFV: SFV_(—)2937-   SSN: SSON_(—)3042 SSON_(—)3489(yhfK)-   SBO: SBO_(—)3103-   SDY: SDY_(—)3193-   ECA: ECA2789-   PLU: plu3987-   ENT: Ent638_(—)3307-   SPE: Spro_(—)2201-   VPA: VPA0278-   VFI: VF0403-   PPR: PBPRA0469(mvaD)-   PEN: PSEEN4850-   CBU: CBU_(—)0607(mvaD)-   CBD: COXBU7E912_(—)0619(mvaD)-   LPN: lpg2051-   LPF: lpl2029-   LPP: lpp2034-   TCX: Tcr_(—)1718-   HHA: Hhal_(—)1623-   DNO: DNO_(—)0798-   EBA: ebA5678 p2A143-   DVU: DVU1679(idi)-   DDE: Dde_(—)1991-   LIP: LI1134-   BBA: Bd1626-   AFW: Anae109_(—)4082-   MXA: MXAN_(—)5021(fni)-   RPR: RP452-   RTY: RT0439(idi)-   RCO: RC0744-   RFE: RF_(—)0785(fni)-   RBE: RBE_(—)0731(fni)-   RAK: A1C_(—)04190-   RBO: A1I_(—)04755-   RCM: A1E_(—)02555-   RRI: A1G_(—)04195-   MLO: mlr6371-   RET: RHE_PD00245(ypd00046)-   XAU: Xaut_(—)4134-   SIL: SP00131-   SIT: TM1040_(—)3442-   RSP: RSP_(—)0276-   RSH: Rsph17029_(—)1919-   RSQ: Rsph17025_(—)1019-   JAN: Jann_(—)0168-   RDE: RD1_(—)0147(idi)-   DSH: Dshi_(—)3527-   BSU: BG11440(ypgA)-   BAN: BA1520-   BAR: GBAA1520-   BAA: BA_(—)2041-   BAT: BAS1409-   BCE: BC1499-   BCA: BCE_(—)1626-   BCZ: BCZK1380(fni)-   BCY: Bcer98_(—)1222-   BTK: BT9727_(—)1381(fni)-   BTL: BALH_(—)1354-   BLI: BL02217(fni)-   BLD: BLi02426-   BAY: RBAM_(—)021020(fni)-   BPU: BPUM_(—)2020(fni)-   OIH: OB0537-   SAU: SA2136(fni)-   SAV: SAV2346(fni)-   SAM: MW2267(fni)-   SAR: SAR2431(fni)-   SAS: SAS2237-   SAC: SACOL2341(fni)-   SAB: SAB2225c(fni)-   SAA: SAUSA300_(—)2292(fni)-   SAO: SAOUHSC_(—)02623-   SEP: SE1925-   SER: SERP1937(fni-2)-   SHA: SH0712(fni)-   SSP: SSP056-   LMO: lmo 1383-   LMF: LMOf2365_(—)1402(fni)-   LIN: lin1420-   LWE: lwe1399(fni)-   LLA: L11083(yebB)-   LLC: LACR_(—)0457-   LLM: llmg_(—)0428(fni)-   SPY: SPy_(—)0879-   SPZ: M5005_Spy_(—)0685-   SPM: spyM18_(—)0940-   SPG: SpyM3_(—)0598-   SPS: SPs1255-   SPH: MGAS10270_Spy0743-   SPI: MGAS10750_Spy0777-   SPJ: MGAS2096_Spy0756-   SPK: MGAS9429_Spy0740-   SPF: SpyM51123(fni)-   SPA: M6_Spy0702-   SPB: M28_Spy0665-   SPN: SP_(—)0384-   SPR: spr0341(fni)-   SPD: SPD_(—)0349(fni)-   SAG: SAG1323-   SAN: gbs1393-   SAK: SAK_(—)1354(fni)-   SMU: SMU.939-   STC: str0562(idi)-   STL: stu0562(idi)-   STE: STER_(—)0601-   SSA: SSA_(—)0336-   SGO: SGO_(—)0242-   LPL: lp_(—)1732(idi1)-   LJO: LJ1208 LAC: LBA1171-   LSA: LSA0905(idi)-   LSL: LSL_(—)0682-   LDB: Ldb0996(fni)-   LBU: LBUL_(—)0903-   LBR: LVIS_(—)0861-   LCA: LSEI_(—)1493-   LGA: LGAS_(—)1036-   LRE: Lreu_(—)0912-   EFA: EF0901-   OOE: OEOE_(—)1103-   STH: STH1674-   CBE: Cbei_(—)3081-   DRM: Dred_(—)0474-   SWO: Swol_(—)1341-   MTA: Moth_(—)1328-   MTU: Rv1745c(idi)-   MTC: MT1787(idi)-   MBO: Mb1774c(idi)-   MBB: BCG_(—)1784c(idi)-   MPA: MAP3079c-   MAV: MAV_(—)3894(fni)-   MSM: MSMEG_(—)1057(fni) MSMEG_(—)2337(fni)-   MUL: MUL_(—)0380(idi2)-   MVA: Mvan_(—)1582 Mvan_(—)2176-   MGI: Mflv_(—)1842 Mflv_(—)4187-   MMC: Mmcs_(—)1954-   MKM: Mkms_(—)2000-   MJL: Mjls_(—)1934-   CGL: NCgl2223(cg12305)-   CGB: cg2531(idi)-   CEF: CE2207-   CDI: DIP1730(idi)-   NFA: nfa19790 nfa22100-   RHA: RHA1_ro00239-   SCO: SCO6750(SC5F2A.33c)-   SMA: SAV1663(idi)-   LXX: Lxx23810(idi)-   CMI: CMM_(—)2889(idiA)-   AAU: AAur_(—)0321(idi)-   PAC: PPA2115-   FRA: Francci3_(—)4188-   FRE: Franean1_(—)5570-   FAL: FRAAL6504(idi)-   KRA: Krad_(—)3991-   SEN: SACE_(—)2627(idiB_(—)2) SACE_(—)5210(idi)-   STP: Strop_(—)4438-   SAQ: Sare_(—)4564 Sare_(—)4928-   RXY: Rxy10400-   BBU: BB0684-   BGA: BG0707-   SYN: sll1556-   SYC: syc2161_c-   SYF: Synpcc7942_(—)1933-   CYA: CYA_(—)2395(fni)-   CYB: CYB_(—)2691(fni)-   TEL: tll1403-   ANA: all4591-   AVA: Ava_(—)2461 Ava_B0346-   TER: Tery_(—)1589-   SRU: SRU_(—)1900(idi)-   CHU: CHU_(—)0674(idi)-   GFO: GFO_(—)2363(idi)-   FJO: Fjoh_(—)0269-   FPS: FP1792(idi)-   CTE: CT0257-   CCH: Cag_(—)1445-   CPH: Cpha266_(—)0385-   PVI: Cvib_(—)1545-   PLT: Plut_(—)1764-   RRS: RoseRS_(—)2437-   RCA: Rcas_(—)2215-   HAU: Haur_(—)4687-   DRA: DR_(—)1087-   DGE: Dgeo_(—)1381-   TTH: TT_P0067-   TTJ: TTHB110-   MJA: MJ0862-   MMP: MMP0043-   MMQ: MmarC5_(—)1637-   MMX: MmarC6_(—)0906-   MMZ: MmarC7_(—)1040-   MAE: Maeo_(—)1184-   MVN: Mevan_(—)1058-   MAC: MA0604(idi)-   MBA: Mbar_A1419-   MMA: MM_(—)1764-   MBU: Mbur_(—)2397-   MTP: Mthe_(—)0474-   MHU: Mhun_(—)2888-   MLA: Mlab_(—)1665-   MEM: Memar_(—)1814-   MBN: Mboo 2211-   MTH: MTH48-   MST: Msp_(—)0856(fni)-   MSI: Msm_(—)1441-   MKA: MK0776(lldD)-   AFU: AF2287-   HAL: VNG1818G(idi) VNG6081G(crt_(—)1) VNG6445G(crt_(—)2) VNG7060    VNG7149-   HMA: rrnAC3484(idi)-   HWA: HQ2772A(idiA) HQ2847A(idiB)-   NPH: NP0360A(idiB_(—)1) NP4826A(idiA) NP5124A(idiB_(—)2)-   TAC: Ta0102-   TVO: TVN0179-   PTO: PT00496-   PHO: PH1202-   PAB: PAB1662-   PFU: PF0856-   TKO: TK1470-   RCI: LRC397(fni)-   APE: APE_(—)1765.1-   SMR: Smar_(—)0822-   IHO: Igni_(—)0804-   HBU: Hbut_(—)0539-   SSO: SS00063-   STO: ST2059-   SAI: Saci_(—)0091-   MSE: Msed_(—)2136-   PAI: PAE0801-   PIS: Pisl_(—)1093-   PCL: Pcal_(—)0017-   PAS: Pars_(—)0051-   TPE: Tpen_(—)0272

Exemplary isoprene synthase nucleic acids and polypeptides

Genbank Accession Nos.

-   AY341431-   AY316691-   AY279379-   AJ457070-   AY182241

What is claimed is:
 1. A polyisoprene polymer which is comprised ofrepeat units that are derived from isoprene monomer, wherein thepolyisoprene polymer has δ¹³C value of greater than −22‰ or which iswithin the range of −30‰ to −28.5‰.
 2. A polyisoprene polymer asspecified in claim 1 wherein the polyisoprene polymer has δ¹³C valuewhich is within the range of −21‰ to −12‰.
 3. A polyisoprene polymer asspecified in claim 1 wherein the polyisoprene polymer has δ¹³C valuewhich is within the range of −29.5% to −28.5‰.
 4. A polyisoprene polymeras specified in claim 1 wherein the polyisoprene polymer has δ¹³C valuewhich is within the range of −30‰ to −29‰.
 5. A polyisoprene polymer asspecified in claim 1 wherein the polyisoprene polymer is a homopolymer.6. A polymer as specified in claim 1 wherein the polymer which iscomprised of repeat units which are derived from isoprene, wherein theisoprene is produced by (a) culturing cells comprising a heterologousnucleic acid encoding an isoprene synthase polypeptide under suitableculture conditions for the production of the isoprene, (b) producing theisoprene, and (c) recovering the isoprene from the culture.
 7. Apolyisoprene polymer as specified in claim 1 wherein the polyisoprenepolymer has a 3,4-microstructure content of greater than 10 percent. 8.A polyisoprene polymer as specified in claim 1 wherein the polyisoprenepolymer has a 1,2-microstructure content of greater than 10 percent. 9.A polyisoprene polymer which is comprised of repeat units that arederived from isoprene monomer, wherein the polyisoprene polymer has δ¹³Cvalue which is within the range of −34‰ to −24‰, and wherein thepolyisoprene (i) has a cis-1,4-microstructure content of less than 99.9%and a trans-1,4-microstructure content of less than 99.9%, or (ii) has a3,4-microstructure content of greater than 2%, or (iii) has a1,2-microstructure content of greater than 2%, or (iv) has weightaverage molecular weight which is within the range of 5,000 to 100,000.10. A polyisoprene polymer as specified in claim 9 wherein thepolyisoprene polymer has δ¹³C value which is within the range of −33‰ to−25‰.
 11. A polyisoprene polymer as specified in claim 10 wherein thepolyisoprene polymer has a 1,2-microstructure content of greater than5%.
 12. A polyisoprene polymer as specified in claim 11 wherein thepolyisoprene polymer has a polydispersity of less than 2.0.
 13. Apolyisoprene polymer as specified in claim 9 wherein the polyisoprenepolymer is a liquid polyisoprene polymer having a weight averagemolecular weight which is within the range of 30,000 to 50,000.
 14. Apolymer as specified in claim 9 wherein the polymer which is comprisedof repeat units which are derived from isoprene, wherein the isoprene isproduced by (a) culturing cells comprising a heterologous nucleic acidencoding an isoprene synthase polypeptide under suitable cultureconditions for the production of the isoprene, (b) producing theisoprene, and (c) recovering the isoprene from the culture.
 15. Apolymer which is comprised of repeat units that are derived fromisoprene monomer and at least one additional monomer, wherein thepolymer includes blocks of repeat units that are derived from isoprene,and wherein the blocks of repeat units that are derived from isoprenehave a δ¹³C value of greater than −22‰ or which is within the range of−34‰ to −24‰.
 16. A polymer as specified in claim 15 wherein the polymeris a copolymer selected from the group consistion of (i) copolymers ofisoprene and 1,3-butadiene, (ii) copolymers of isoprene and styrene,(iii) copolymers of isoprene, 1,3-butadiene, and styrene, and (iv)copolymers of isoprene and α-methyl styrene.
 17. A polyisoprene polymeras specified in claim 13 wherein the liquid polyisoprene polymer has apolydispersity of less than 1.8.
 18. A method for verifying that apolyisoprene homopolymer is from a sustainable renewable non-petroleumderived source which comprises: (I) determining the δ¹³C value of thepolyisoprene homopolymer; (II) if the polyisoprene homopolymer has aδ¹³C value within the range of −34‰ to −30‰ or within the range of−28.5‰ to −24‰ additionally analyzing the polyisoprene homopolymer todetermine (1) its cis-microstructure content, (2) its 3,4-microstructurecontent, (3) its 1,2-microstructure content, (4) its a weight averagemolecular weight, or (5) the presence or absence of residual proteins,soaps, lipids, resins, or sugars indicative of natural rubber; and (III)verifying that the polyisoprene homopolymer is from a sustainablerenewable non-petroleum derived source if it has (i) a δ¹³C value ofgreater than −22‰, (ii) a δ¹³C value which is within the range of −30‰to −28.5‰, or (iii) a δ¹³C value within the range of −34‰ to −30‰ orwithin the range of −28.5% to −24‰ and if it (a) has acis-microstructure content of less than 100%, (b) contains3,4-microstructure, (c) contains 1,2-microstructure, (d) has a weightaverage molecular weight of less than 100,000, or (e) is free ofresidual proteins, soaps, lipids, resins, or sugars indicative ofnatural rubber.
 19. A method as specified in claim 18 wherein theverification is made by showing that the polyisoprene homopolymer has aδ¹³C value which is within the range of −34‰ to −30‰ and by showing thatthe polyisoprene homopolymer is free of residual proteins, soaps,lipids, resins, or sugars indicative of natural rubber.
 20. A method forverifying that a copolymer having repeat units that are derived fromisoprene contains isoprene that is from a sustainable renewablenon-petroleum derived source, said method comprising: (I) determiningthe δ¹³C value of at least one polyisoprene block in the copolymer; and(II) verifying that the isoprene in the copolymer is from a sustainablerenewable non-petroleum derived source if the polyisoprene block has (i)a δ¹³C value of greater than −22‰, or (ii) a δ¹³C value which is withinthe range of −34‰ to −28.5.